Polypeptides Having Cellobiohydrolase 1 Activity and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to polypeptides having cellobiohydrolase I activity and polynucleotides having a nucleotide sequence which encodes for the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid constructs as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/646,980filed on Oct. 8, 2012, now pending, which is a divisional of U.S.application Ser. No. 13/483,389 filed on May 30, 2012, now pending,which is a divisional of U.S. application Ser. No. 12/818,861 filed onJun. 18, 2010, now allowed, which is a continuation of U.S. applicationSer. No. 10/481,179 filed Dec. 17, 2003, now U.S. Pat. No. 7,785,853,which is a 35 U.S.C. 371 national application of internationalapplication no. PCT/DK02/000429 filed Jun. 26, 2002, which claimspriority or the benefit under 35 U.S.C. 119 of Danish application no. PA2001 01000 filed on Jun. 26, 2001, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to polypeptides having cellobiohydrolase I(also referred to as CBH I or CBH 1) activity and polynucleotides havinga nucleotide sequence which encodes for the polypeptides. The inventionalso relates to nucleic acid constructs, vectors, and host cellscomprising the nucleic acid constructs as well as methods for producingand using the polypeptides.

BACKGROUND OF THE INVENTION

Cellulose is an important industrial raw material and a source ofrenewable energy. The physical structure and morphology of nativecellulose are complex and the fine details of its structure have beendifficult to determine experimentally. However, the chemical compositionof cellulose is simple, consisting of D-glucose residues linked bybeta-1,4-glycosidic bonds to form linear polymers with chains length ofover 10,000 glycosidic residues.

In order to be efficient, the digestion of cellulose requires severaltypes of enzymes acting cooperatively. At least three categories ofenzymes are necessary to convert cellulose into glucose: endo(1,4)-beta-D-glucanases (EC 3.2.1.4) that cut the cellulose chains atrandom; cellobiohydrolases (EC 3.2.1.91) which cleave cellobiosyl unitsfrom the cellulose chain ends and beta-glucosidases (EC 3.2.1.21) thatconvert cellobiose and soluble cellodextrins into glucose. Among thesethree categories of enzymes involved in the biodegradation of cellulose,cellobiohydrolases are the key enzymes for the degradation of nativecrystalline cellulose.

Exo-cellobiohydrolases (Cellobiohydrolase I, or CBH I) refer to thecellobiohydrolases which degrade cellulose by hydrolyzing the cellobiosefrom the reducing end of the cellulose polymer chains.

It is an object of the present invention to provide improvedpolypeptides having cellobiohydrolase I activity and polynucleotidesencoding the polypeptides. The improved polypeptides may have improvedspecific activity and/or improved stability—in particular improvedthermostability. The polypeptides may also have an improved ability toresist inhibition by cellobiose.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a polypeptide havingcellobiohydrolase I activity, selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of: an amino acid sequence which has at least 80%identity with amino acids 1 to 526 of SEQ ID NO:2,

an amino acid sequence which has at least 80% identity with amino acids1 to 529 of SEQ ID NO:4,

an amino acid sequence which has at least 80% identity with amino acids1 to 451 of SEQ ID NO:6,

an amino acid sequence which has at least 80% identity with amino acids1 to 457 of SEQ ID NO:8,

an amino acid sequence which has at least 80% identity with amino acids1 to 538 of SEQ ID NO:10,

an amino acid sequence which has at least 70% identity with amino acids1 to 415 of SEQ ID NO:12,

an amino acid sequence which has at least 70% identity with amino acids1 to 447 of SEQ ID NO:14,

an amino acid sequence which has at least 80% identity with amino acids1 to 452 of SEQ ID NO:16,

an amino acid sequence which has at least 80% identity with amino acids1 to 454 of SEQ ID NO:38,

an amino acid sequence which has at least 80% identity with amino acids1 to 458 of SEQ ID NO:40,

an amino acid sequence which has at least 80% identity with amino acids1 to 450 of SEQ ID NO:42,

an amino acid sequence which has at least 80% identity with amino acids1 to 446 of SEQ ID NO:44,

an amino acid sequence which has at least 80% identity with amino acids1 to 527 of SEQ ID NO:46,

an amino acid sequence which has at least 80% identity with amino acids1 to 455 of SEQ ID NO:48,

an amino acid sequence which has at least 80% identity with amino acids1 to 464 of SEQ ID NO:50,

an amino acid sequence which has at least 80% identity with amino acids1 to 460 of SEQ ID NO:52,

an amino acid sequence which has at least 80% identity with amino acids1 to 450 of SEQ ID NO:54,

an amino acid sequence which has at least 80% identity with amino acids1 to 532 of SEQ ID NO:56,

an amino acid sequence which has at least 80% identity with amino acids1 to 460 of SEQ ID NO:58,

an amino acid sequence which has at least 80% identity with amino acids1 to 525 of SEQ ID NO:60, and

an amino acid sequence which has at least 80% identity with amino acids1 to 456 of SEQ ID NO:66;

(b) a polypeptide comprising an amino acid sequence selected from thegroup consisting of:

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Acremonium thermophilum,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Chaetomium thermophilum,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Scytalidium sp.,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Scytalidium thermophilum,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Thermoascus aurantiacus,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Thielavia australiensis,

an amino acid sequence which has at least 70% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Verticillium tenerum,

an amino acid sequence which has at least 70% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Neotermes castaneus,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Melanocarpus albomyces,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Acremonium sp.,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Chaetomidium pingtungium,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Sporotrichum pruinosum,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Diplodia gossypina,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Trichophaea saccata,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Myceliophthora thermophila,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Exidia glandulosa,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Xylaria hypoxylon,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Poitrasia circinans,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Coprinus cinereus,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by the cellobiohydrolase I encoding part of thenucleotide sequence present in Pseudoplectania nigrella,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Trichothecium roseum IFO 5372,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Humicola nigrescens CBS 819.73,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Cladorrhinum foecundissimum CBS427.97,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Diplodia gossypina CBS 247.96,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Myceliophthora thermophilaCBS117.65,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Rhizomucor pusillus CBS109471,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Meripilus giganteus CBS 521.95,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Exidia glandulosa CBS 2377.96,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Xylaria hypoxylon CBS 284.96,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Trichophaea saccata CBS 804.70,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Chaetomium sp.,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Myceliophthora hinnulea,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Thielavia cf. microspora,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Aspergillus sp.,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Scopulariopsis sp.,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Fusarium sp.,

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Verticillium sp., and

an amino acid sequence encoded by the cellobiohydrolase I encoding partof the nucleotide sequence present in Phytophthora infestans;

(c) a polypeptide comprising an amino acid sequence selected from thegroup consisting of:

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1578 of SEQ ID NO:1,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1587 of SEQ ID NO:3,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1353 of SEQ ID NO:5,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1371 of SEQ ID NO:7,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1614 of SEQ ID NO:9,

an amino acid sequence which has at least 70% identity with thepolypeptide encoded by nucleotides 1 to 1245 of SEQ ID NO:11,

an amino acid sequence which has at least 70% identity with thepolypeptide encoded by nucleotides 1 to 1341 of SEQ ID NO:13,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1356 of SEQ ID NO:15,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1365 of SEQ ID NO:37,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1377 of SEQ ID NO:39,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1353 of SEQ ID NO:41,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1341 of SEQ ID NO:43,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1584 of SEQ ID NO:45,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1368 of SEQ ID NO:47,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1395 of SEQ ID NO:49,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1383 of SEQ ID NO:51,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1353 of SEQ ID NO:53,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1599 of SEQ ID NO:55,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1383 of SEQ ID NO:57,

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1578 of SEQ ID NO:59, and

an amino acid sequence which has at least 80% identity with thepolypeptide encoded by nucleotides 1 to 1371 of SEQ ID NO:65;

(d) a polypeptide which is encoded by a nucleotide sequence whichhybridizes under high stringency conditions with a polynucleotide probeselected from the group consisting of:

(i) the complementary strand of the nucleotides selected from the groupconsisting of:

nucleotides 1 to 1578 of SEQ ID NO:1,

nucleotides 1 to 1587 of SEQ ID NO:3,

nucleotides 1 to 1353 of SEQ ID NO:5,

nucleotides 1 to 1371 of SEQ ID NO:7,

nucleotides 1 to 1614 of SEQ ID NO:9,

nucleotides 1 to 1245 of SEQ ID NO:11,

nucleotides 1 to 1341 of SEQ ID NO:13,

nucleotides 1 to 1356 of SEQ ID NO:15,

nucleotides 1 to 1365 of SEQ ID NO:37,

nucleotides 1 to 1377 of SEQ ID NO:39,

nucleotides 1 to 1353 of SEQ ID NO:41,

nucleotides 1 to 1341 of SEQ ID NO:43,

nucleotides 1 to 1584 of SEQ ID NO:45,

nucleotides 1 to 1368 of SEQ ID NO:47,

nucleotides 1 to 1395 of SEQ ID NO:49,

nucleotides 1 to 1383 of SEQ ID NO:51,

nucleotides 1 to 1353 of SEQ ID NO:53,

nucleotides 1 to 1599 of SEQ ID NO:55,

nucleotides 1 to 1383 of SEQ ID NO:57,

nucleotides 1 to 1578 of SEQ ID NO:59, and

nucleotides 1 to 1371 of SEQ ID NO:65;

(ii) the complementary strand of the nucleotides selected from the groupconsisting of:

nucleotides 1 to 500 of SEQ ID NO:1,

nucleotides 1 to 500 of SEQ ID NO:3,

nucleotides 1 to 500 of SEQ ID NO:5,

nucleotides 1 to 500 of SEQ ID NO:7,

nucleotides 1 to 500 of SEQ ID NO:9,

nucleotides 1 to 500 of SEQ ID NO:11,

nucleotides 1 to 500 of SEQ ID NO:13,

nucleotides 1 to 500 of SEQ ID NO:15,

nucleotides 1 to 500 of SEQ ID NO:37,

nucleotides 1 to 500 of SEQ ID NO:39,

nucleotides 1 to 500 of SEQ ID NO:41,

nucleotides 1 to 500 of SEQ ID NO:43,

nucleotides 1 to 500 of SEQ ID NO:45,

nucleotides 1 to 500 of SEQ ID NO:47,

nucleotides 1 to 500 of SEQ ID NO:49,

nucleotides 1 to 500 of SEQ ID NO:51,

nucleotides 1 to 500 of SEQ ID NO:53,

nucleotides 1 to 500 of SEQ ID NO:55,

nucleotides 1 to 500 of SEQ ID NO:57,

nucleotides 1 to 500 of SEQ ID NO:59,

nucleotides 1 to 500 of SEQ ID NO:65,

nucleotides 1 to 221 of SEQ ID NO:17,

nucleotides 1 to 239 of SEQ ID NO:18,

nucleotides 1 to 199 of SEQ ID NO:19,

nucleotides 1 to 191 of SEQ ID NO:20,

nucleotides 1 to 232 of SEQ ID NO:21,

nucleotides 1 to 467 of SEQ ID NO:22,

nucleotides 1 to 534 of SEQ ID NO:23,

nucleotides 1 to 563 of SEQ ID NO:24,

nucleotides 1 to 218 of SEQ ID NO:25,

nucleotides 1 to 492 of SEQ ID NO:26,

nucleotides 1 to 481 of SEQ ID NO:27,

nucleotides 1 to 463 of SEQ ID NO:28,

nucleotides 1 to 513 of SEQ ID NO:29,

nucleotides 1 to 579 of SEQ ID NO:30,

nucleotides 1 to 514 of SEQ ID NO:31,

nucleotides 1 to 477 of SEQ ID NO:32,

nucleotides 1 to 500 of SEQ ID NO:33,

nucleotides 1 to 470 of SEQ ID NO:34,

nucleotides 1 to 491 of SEQ ID NO:35,

nucleotides 1 to 221 of SEQ ID NO:36,

nucleotides 1 to 519 of SEQ ID NO:61,

nucleotides 1 to 497 of SEQ ID NO:62,

nucleotides 1 to 498 of SEQ ID NO:63,

nucleotides 1 to 525 of SEQ ID NO:64, and

nucleotides 1 to 951 of SEQ ID NO:67; and

(iii) the complementary strand of the nucleotides selected from thegroup consisting of:

nucleotides 1 to 200 of SEQ ID NO:1,

nucleotides 1 to 200 of SEQ ID NO:3,

nucleotides 1 to 200 of SEQ ID NO:5,

nucleotides 1 to 200 of SEQ ID NO:7,

nucleotides 1 to 200 of SEQ ID NO:9,

nucleotides 1 to 200 of SEQ ID NO:11,

nucleotides 1 to 200 of SEQ ID NO:13,

nucleotides 1 to 200 of SEQ ID NO:15,

nucleotides 1 to 200 of SEQ ID NO:37,

nucleotides 1 to 200 of SEQ ID NO:39,

nucleotides 1 to 200 of SEQ ID NO:41,

nucleotides 1 to 200 of SEQ ID NO:43,

nucleotides 1 to 200 of SEQ ID NO:45,

nucleotides 1 to 200 of SEQ ID NO:47,

nucleotides 1 to 200 of SEQ ID NO:49,

nucleotides 1 to 200 of SEQ ID NO:51,

nucleotides 1 to 200 of SEQ ID NO:53,

nucleotides 1 to 200 of SEQ ID NO:55,

nucleotides 1 to 200 of SEQ ID NO:57,

nucleotides 1 to 200 of SEQ ID NO:59, and

nucleotides 1 to 200 of SEQ ID NO:65; and

(e) a fragment of (a), (b) or (c) that has cellobiohydrolase I activity.

In a second aspect the present invention relates to a polynucleotidehaving a nucleotide sequence which encodes for the polypeptide of theinvention.

In a third aspect the present invention relates to a nucleic acidconstruct comprising the nucleotide sequence, which encodes for thepolypeptide of the invention, operably linked to one or more controlsequences that direct the production of the polypeptide in a suitablehost.

In a fourth aspect the present invention relates to a recombinantexpression vector comprising the nucleic acid construct of theinvention.

In a fifth aspect the present invention relates to a recombinant hostcell comprising the nucleic acid construct of the invention.

In a sixth aspect the present invention relates to a method forproducing a polypeptide of the invention, the method comprising:

(a) cultivating a strain, which in its wild-type form is capable ofproducing the polypeptide, to produce the polypeptide; and

(b) recovering the polypeptide.

In a seventh aspect the present invention relates to a method forproducing a polypeptide of the invention, the method comprising:

(a) cultivating a recombinant host cell of the invention underconditions conducive for production of the polypeptide; and

(b) recovering the polypeptide.

In an eight aspect the present invention relates to a method for in-situproduction of a polypeptide of the invention, the method comprising:

(a) cultivating a recombinant host cell of the invention underconditions conducive for production of the polypeptide; and

(b) contacting the polypeptide with a desired substrate without priorrecovery of the polypeptide.

Other aspects of the present invention will be apparent from the belowdescription and from the appended claims.

DEFINITIONS

Prior to discussing the present invention in further details, thefollowing terms and conventions will first be defined:

Substantially pure polypeptide: In the present context, the term“substantially pure polypeptide” means a polypeptide preparation whichcontains at the most 10% by weight of other polypeptide material withwhich it is natively associated (lower percentages of other polypeptidematerial are preferred, e.g., at the most 8% by weight, at the most 6%by weight, at the most 5% by weight, at the most 4% at the most 3% byweight, at the most 2% by weight, at the most 1% by weight, and at themost ½% by weight). Thus, it is preferred that the substantially purepolypeptide is at least 92% pure, i.e., that the polypeptide constitutesat least 92% by weight of the total polypeptide material present in thepreparation, and higher percentages are preferred such as at least 94%pure, at least 95% pure, at least 96% pure, at least 96% pure, at least97% pure, at least 98% pure, at least 99%, and at the most 99.5% pure.The polypeptides disclosed herein are preferably in a substantially pureform. In particular, it is preferred that the polypeptides disclosedherein are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods.Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form”.

Cellobiohydrolase I activity: The term “cellobiohydrolase I activity” isdefined herein as a cellulose 1,4-beta-cellobiosidase (also referred toas Exo-glucanase, Exo-cellobiohydrolase or 1,4-beta-cellobiohydrolase)activity, as defined in the enzyme class EC 3.2.1.91, which catalyzesthe hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose andcellotetraose, releasing cellobiose from the reducing ends of thechains.

For purposes of the present invention, cellobiohydrolase I activity maybe determined according to the procedure described in Example 2.

In an embodiment, cellobiohydrolase I activity may be determinedaccording to the procedure described in Deshpande et al., Methods inEnzymology, pp. 126-130 (1988): “Selective Assay forExo-1,4-Beta-Glucanases”. According to this procedure, one unit ofcellobiohydrolase I activity (agluconic bond cleavage activity) isdefined as 1.0 micromole of p-nitrophenol produced per minute at 50° C.,pH 5.0.

The polypeptides of the present invention should preferably have atleast 20% of the cellobiohydrolase I activity of a polypeptideconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQID NO:66. In a particular preferred embodiment, the polypeptides shouldhave at least 40%, such as at least 50%, preferably at least 60%, suchas at least 70%, more preferably at least 80%, such as at least 90%,most preferably at least 95%, such as about or at least 100% of thecellobiohydrolase I activity of the polypeptide consisting of the aminoacid sequence selected from the group consisting of amino acids 1 to 526of SEQ ID NO:2, amino acids 1 to 529 of SEQ ID NO:4, amino acids 1 to451 of SEQ ID NO:6, amino acids 1 to 457 of SEQ ID NO:8, amino acids 1to 538 of SEQ ID NO:10, amino acids 1 to 415 of SEQ ID NO:12, aminoacids 1 to 447 of SEQ ID NO:14, amino acids 1 to 452 of SEQ ID NO:16,amino acids 1 to 454 of SEQ ID NO:38, amino acids 1 to 458 of SEQ IDNO:40, amino acids 1 to 450 of SEQ ID NO:42, amino acids 1 to 446 of SEQID NO:44, amino acids 1 to 527 of SEQ ID NO:46, amino acids 1 to 455 ofSEQ ID NO:48, amino acids 1 to 464 of SEQ ID NO:50, amino acids 1 to 460of SEQ ID NO:52, amino acids 1 to 450 of SEQ ID NO:54, amino acids 1 to532 of SEQ ID NO:56, amino acids 1 to 460 of SEQ ID NO:58, amino acids 1to 525 of SEQ ID NO:60, and amino acids 1 to 456 of SEQ ID NO:66.

Identity: In the present context, the homology between two amino acidsequences or between two nucleotide sequences is described by theparameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by using the program FASTAincluded in version 2.0× of the FASTA program package (see Pearson andLipman, 1988, “Improved Tools for Biological Sequence Analysis”, PNAS85:2444-2448; and Pearson, 1990, “Rapid and Sensitive SequenceComparison with FASTP and FASTA”, Methods in Enzymology 183:63-98). Thescoring matrix used was BLOSUM50, gap penalty was −12, and gap extensionpenalty was −2.

The degree of identity between two nucleotide sequences is determinedusing the same algorithm and software package as described above. Thescoring matrix used was the identity matrix, gap penalty was −16, andgap extension penalty was −4.

Fragment: When used herein, a “fragment” of a sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38,SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,SEQ ID NO:60, and SEQ ID NO:66 is a polypeptide having one or more aminoacids deleted from the amino and/or carboxyl terminus of this amino acidsequence. Preferably, a fragment is a polypeptide having the amino acidsequence deleted corresponding to the “cellulose-binding domain” and/orthe “linker domain” of Trichoderma reesei cellobiohydrolase I asdescribed in SWISS-PROT accession number P00725. More preferably, afragment comprises the amino acid sequence corresponding to the“catalytic domain” of Trichoderma reesei cellobiohydrolase I asdescribed in SWISS-PROT accession number P00725. Most preferably, afragment contains at least 434 amino acid residues, e.g., the amino acidresidues selected from the group consisting of amino acids 1 to 434 ofSEQ ID NO:2, amino acids 1 to 434 of SEQ ID NO:4, amino acids 1 to 434of SEQ ID NO:6, amino acids 1 to 434 of SEQ ID NO:8, amino acids 1 to434 of SEQ ID NO:10, amino acids 1 to 434 of SEQ ID NO:14, amino acids 1to 434 of SEQ ID NO:16, amino acids 1 to 434 of SEQ ID NO:38, aminoacids 1 to 434 of SEQ ID NO:40, amino acids 1 to 434 of SEQ ID NO:42,amino acids 1 to 434 of SEQ ID NO:44, amino acids 1 to 434 of SEQ IDNO:46, amino acids 1 to 434 of SEQ ID NO:48, amino acids 1 to 434 of SEQID NO:50, amino acids 1 to 434 of SEQ ID NO:52, amino acids 1 to 434 ofSEQ ID NO:54, amino acids 1 to 434 of SEQ ID NO:56, amino acids 1 to 434of SEQ ID NO:58, amino acids 1 to 434 of SEQ ID NO:60, and amino acids 1to 434 of SEQ ID NO:66. In particular, a fragment contains at least 215amino acid residues, e.g., the amino acid residues selected from thegroup consisting of amino acids 200 to 434 of SEQ ID NO:2, amino acids200 to 434 of SEQ ID NO:4, amino acids 200 to 434 of SEQ ID NO:6, aminoacids 200 to 434 of SEQ ID NO:8, amino acids 200 to 434 of SEQ ID NO:10,amino acids 200 to 415 of SEQ ID NO:12, amino acids 200 to 434 of SEQ IDNO:14, amino acids 200 to 434 of SEQ ID NO:16, amino acids 200 to 434 ofSEQ ID NO:38, amino acids 200 to 434 of SEQ ID NO:40, amino acids 200 to434 of SEQ ID NO:42, amino acids 200 to 434 of SEQ ID NO:44, amino acids200 to 434 of SEQ ID NO:46, amino acids 200 to 434 of SEQ ID NO:48,amino acids 200 to 434 of SEQ ID NO:50, amino acids 200 to 434 of SEQ IDNO:52, amino acids 200 to 434 of SEQ ID NO:54, amino acids 200 to 434 ofSEQ ID NO:56, amino acids 200 to 434 of SEQ ID NO:58, amino acids 200 to434 of SEQ ID NO:60, and amino acids 200 to 434 of SEQ ID NO:66.

Allelic variant: In the present context, the term “allelic variant”denotes any of two or more alternative forms of a gene occupying thesame chromosomal locus. Allelic variation arises naturally throughmutation, and may result in polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or mayencode polypeptides having altered amino acid sequences. An allelicvariant of a polypeptide is a polypeptide encoded by an allelic variantof a gene.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparation,wherein the polynucleotide has been removed from its natural geneticmilieu, and is thus free of other extraneous or unwanted codingsequences and is in a form suitable for use within geneticallyengineered protein production systems. Thus, a substantially purepolynucleotide contains at the most 10% by weight of otherpolynucleotide material with which it is natively associated (lowerpercentages of other polynucleotide material are preferred, e.g., at themost 8% by weight, at the most 6% by weight, at the most 5% by weight,at the most 4% at the most 3% by weight, at the most 2% by weight, atthe most 1% by weight, and at the most ½% by weight). A substantiallypure polynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 92% pure, i.e.,that the polynucleotide constitutes at least 92% by weight of the totalpolynucleotide material present in the preparation, and higherpercentages are preferred such as at least 94% pure, at least 95% pure,at least 96% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, and at the most 99.5% pure. The polynucleotidesdisclosed herein are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form”.

Modification(s): In the context of the present invention the term“modification(s)” is intended to mean any chemical modification of apolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, and SEQ ID NO:66, as well as genetic manipulation of the DNAencoding that polypeptide. The modification(s) can be replacement(s) ofthe amino acid side chain(s), substitution(s), deletion(s) and/orinsertions(s) in or at the amino acid(s) of interest.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having cellobiohydrolase I activity, which has beenproduced by an organism which is expressing a modified gene as comparedto SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, or SEQ IDNO:65. The modified gene, from which said variant is produced whenexpressed in a suitable host, is obtained through human intervention bymodification of a nucleotide sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, and SEQID NO:65.

cDNA: The term “cDNA” when used in the present context, is intended tocover a DNA molecule which can be prepared by reverse transcription froma mature, spliced, mRNA molecule derived from a eukaryotic cell. cDNAlacks the intron sequences that are usually present in the correspondinggenomic DNA. The initial, primary RNA transcript is a precursor to mRNAand it goes through a series of processing events before appearing asmature spliced mRNA. These events include the removal of intronsequences by a process called splicing. When cDNA is derived from mRNAit therefore lacks intron sequences.

Nucleic acid construct: When used herein, the term “nucleic acidconstruct” means a nucleic acid molecule, either single- ordouble-stranded, which is isolated from a naturally occurring gene orwhich has been modified to contain segments of nucleic acids in a mannerthat would not otherwise exist in nature. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polypeptide of the present invention. Each controlsequence may be native or foreign to the nucleotide sequence encodingthe polypeptide. Such control sequences include, but are not limited to,a leader, polyadenylation sequence, propeptide sequence, promoter,signal peptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

Operably linked: The term “operably linked” is defined herein as aconfiguration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the expression of a polypeptide.

Coding sequence: When used herein the term “coding sequence” is intendedto cover a nucleotide sequence, which directly specifies the amino acidsequence of its protein product. The boundaries of the coding sequenceare generally determined by an open reading frame, which usually beginswith the ATG start codon. The coding sequence typically include DNA,cDNA, and recombinant nucleotide sequences.

Expression: In the present context, the term “expression” includes anystep involved in the production of the polypeptide including, but notlimited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.

Expression vector: In the present context, the term “expression vector”covers a DNA molecule, linear or circular, that comprises a segmentencoding a polypeptide of the invention, and which is operably linked toadditional segments that provide for its transcription.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation with a nucleic acid construct.

The terms “polynucleotide probe”, “hybridization” as well as the variousstringency conditions are defined in the section entitled “PolypeptidesHaving Cellobiohydrolase I Activity”.

Thermostability: The term “thermostability”, as used herein, is measuredas described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides HavingCellobiohydrolase I Activity

In a first embodiment, the present invention relates to polypeptideshaving cellobiohydrolase I activity and where the polypeptidescomprises, preferably consists of, an amino acid sequence which has adegree of identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, and SEQ ID NO:66 (i.e., the mature polypeptide) of at least 65%,preferably at least 70%, e.g., at least 75%, more preferably at least80%, such as at least 85%, even more preferably at least 90%, mostpreferably at least 95%, e.g., at least 96%, such as at least 97%, andeven most preferably at least 98%, such as at least 99% (hereinafter“homologous polypeptides”). In an interesting embodiment, the amino acidsequence differs by at the most ten amino acids (e.g., by ten aminoacids), in particular by at the most five amino acids (e.g., by fiveamino acids), such as by at the most four amino acids (e.g., by fouramino acids), e.g., by at the most three amino acids (e.g., by threeamino acids) from an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, and SEQ ID NO:66. In a particular interesting embodiment, theamino acid sequence differs by at the most two amino acids (e.g., by twoamino acids), such as by one amino acid from an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:66.

Preferably, the polypeptides of the present invention comprise an aminoacid sequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:66; anallelic variant thereof; or a fragment thereof that hascellobiohydrolase I activity. In another preferred embodiment, thepolypeptide of the present invention consists of an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:66.

The polypeptide of the invention may be a wild-type cellobiohydrolase Iidentified and isolated from a natural source. Such wild-typepolypeptides may be specifically screened for by standard techniquesknown in the art, such as molecular screening as described in Example 1.Furthermore, the polypeptide of the invention may be prepared by the DNAshuffling technique, such as described in Ness et al., NatureBiotechnology 17: 893-896 (1999). Moreover, the polypeptide of theinvention may be an artificial variant which comprises, preferablyconsists of, an amino acid sequence that has at least one substitution,deletion and/or insertion of an amino acid as compared to an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:66. Such artificialvariants may be constructed by standard techniques known in the art,such as by site-directed/random mutagenesis of the polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQID NO:66. In one embodiment of the invention, amino acid changes (in theartificial variant as well as in wild-type polypeptides) are of a minornature, that is conservative amino acid substitutions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine, valine andmethionine), aromatic amino acids (phenylalanine, tryptophan andtyrosine), and small amino acids (glycine, alanine, serine andthreonine). Amino acid substitutions which do not generally alter thespecific activity are known in the art and are described, for example,by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press,New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly aswell as these in reverse.

In an interesting embodiment of the invention, the amino acid changesare of such a nature that the physico-chemical properties of thepolypeptides are altered. For example, amino acid changes may beperformed, which improve the thermal stability of the polypeptide, whichalter the substrate specificity, which changes the pH optimum, and thelike.

Preferably, the number of such substitutions, deletions and/orinsertions as compared to an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, and SEQ ID NO:66 is at the most 10, such as at the most 9, e.g.,at the most 8, more preferably at the most 7, e.g., at the most 6, suchas at the most 5, most preferably at the most 4, e.g., at the most 3,such as at the most 2, in particular at the most 1.

The present inventors have isolated nucleotide sequences encodingpolypeptides having cellobiohydrolase I activity from the microorganismsselected from the group consisting of Acremonium thermophilum,Chaetomium thermophilum, Scytalidium sp., Scytalidium thermophilum,Thermoascus aurantiacus, Thielavia australiensis, Verticillium tenerum,Melanocarpus albomyces, Poitrasia circinans, Coprinus cinereus,Trichothecium roseum, Humicola nigrescens, Cladorrhinum foecundissimum,Diplodia gossypina, Myceliophthora thermophila, Rhizomucor pusillus,Meripilus giganteus, Exidia glandulosa, Xylaria hypoxylon, Trichophaeasaccata, Acremonium sp., Chaetomium sp., Chaetomidium pingtungium,Myceliophthora thermophila, Myceliophthora hinnulea, Sporotrichumpruinosum, Thielavia cf. microspora, Aspergillus sp., Scopulariopsissp., Fusarium sp., Verticillium sp., Pseudoplectania nigrella, andPhytophthora infestans; and from the gut of the termite larvae Neotermescastaneus. Thus, in a second embodiment, the present invention relatesto polypeptides comprising an amino acid sequence which has at least 65%identity with the polypeptide encoded by the cellobiohydrolase Iencoding part of the nucleotide sequence present in an organism selectedfrom the group consisting of Acremonium thermophilum, Chaetomiumthermophilum, Scytalidium sp., Scytalidium thermophilum, Thermoascusaurantiacus, Thielavia australiensis, Verticillium tenerum, Neotermescastaneus, Melanocarpus albomyces, Poitrasia circinans, Coprinuscinereus, Trichothecium roseum IFO 5372, Humicola nigrescens CBS 819.73,Cladorrhinum foecundissimum CBS 427.97, Diplodia gossypina CBS 247.96,Myceliophthora thermophila CBS117.65, Rhizomucor pusillus CBS109471,Meripilus giganteus CBS 521.95, Exidia glandulosa CBS 2377.96, Xylariahypoxylon CBS 284.96, Trichophaea saccata CBS 804.70, Acremonium sp.,Chaetomium sp., Chaetomidium pingtungium, Myceliophthora thermophila,Myceliophthora hinnulea, Sporotrichum pruinosum, Thielavia cf.microspora, Aspergillus sp., Scopulariopsis sp., Fusarium sp.,Verticillium sp., Pseudoplectania nigrella, and Phytophthora infestans.In an interesting embodiment of the invention, the polypeptide comprisesan amino acid sequence which has at least 70%, e.g., at least 75%,preferably at least 80%, such as at least 85%, more preferably at least90%, most preferably at least 95%, e.g., at least 96%, such as at least97%, and even most preferably at least 98%, such as at least 99%identity with the polypeptide encoded by the cellobiohydrolase Iencoding part of the nucleotide sequence present in an organism selectedfrom the group consisting of Acremonium thermophilum, Chaetomiumthermophilum, Scytalidium sp., Scytalidium thermophilum, Thermoascusaurantiacus, Thielavia australiensis, Verticillium tenerum, Neotermescastaneus, Melanocarpus albomyces, Poitrasia circinans, Coprinuscinereus, Trichothecium roseum IFO 5372, Humicola nigrescens CBS 819.73,Cladorrhinum foecundissimum CBS 427.97, Diplodia gossypina CBS 247.96,Myceliophthora thermophila CBS117.65, Rhizomucor pusillus CBS109471,Meripilus giganteus CBS 521.95, Exidia glandulosa CBS 2377.96, Xylariahypoxylon CBS 284.96, Trichophaea saccata CBS 804.70, Acremonium sp.,Chaetomium sp., Chaetomidium pingtungium, Myceliophthora thermophila,Myceliophthora hinnulea, Sporotrichum pruinosum, Thielavia cf.microspora, Aspergillus sp., Scopulariopsis sp., Fusarium sp.,Verticillium sp., Pseudoplectania nigrella, and Phytophthora infestans(hereinafter “homologous polypeptides”). In an interesting embodiment,the amino acid sequence differs by at the most ten amino acids (e.g., byten amino acids), in particular by at the most five amino acids (e.g.,by five amino acids), such as by at the most four amino acids (e.g., byfour amino acids), e.g., by at the most three amino acids (e.g., bythree amino acids) from the polypeptide encoded by the cellobiohydrolaseI encoding part of the nucleotide sequence present in an organismselected from the group consisting of Acremonium thermophilum,Chaetomium thermophilum, Scytalidium sp., Scytalidium thermophilum,Thermoascus aurantiacus, Thielavia australiensis, Verticillium tenerum,Neotermes castaneus, Melanocarpus albomyces, Poitrasia circinans,Coprinus cinereus, Trichothecium roseum IFO 5372, Humicola nigrescensCBS 819.73, Cladorrhinum foecundissimum CBS 427.97, Diplodia gossypinaCBS 247.96, Myceliophthora thermophila CBS117.65, Rhizomucor pusillusCBS 109471, Meripilus giganteus CBS 521.95, Exidia glandulosa CBS2377.96, Xylaria hypoxylon CBS 284.96, Trichophaea saccata CBS 804.70,Acremonium sp., Chaetomium sp., Chaetomidium pingtungium, Myceliophthorathermophila, Myceliophthora hinnulea, Sporotrichum pruinosum, Thielaviacf. microspora, Aspergillus sp., Scopulariopsis sp., Fusarium sp.,Verticillium sp., Pseudoplectania nigrella, and Phytophthora infestans.In a particular interesting embodiment, the amino acid sequence differsby at the most two amino acids (e.g., by two amino acids), such as byone amino acid from the polypeptide encoded by the cellobiohydrolase Iencoding part of the nucleotide sequence present in an organism selectedfrom the group consisting of Acremonium thermophilum, Chaetomiumthermophilum, Scytalidium sp., Scytalidium thermophilum, Thermoascusaurantiacus, Thielavia australiensis, Verticillium tenerum, Neotermescastaneus, Melanocarpus albomyces, Poitrasia circinans, Coprinuscinereus, Trichothecium roseum IFO 5372, Humicola nigrescens CBS 819.73,Cladorrhinum foecundissimum CBS 427.97, Diplodia gossypina CBS 247.96,Myceliophthora thermophila CBS117.65, Rhizomucor pusillus CBS109471,Meripilus giganteus CBS 521.95, Exidia glandulosa CBS 2377.96, Xylariahypoxylon CBS 284.96, Trichophaea saccata CBS 804.70, Acremonium sp.,Chaetomium sp., Chaetomidium pingtungium, Myceliophthora thermophila,Myceliophthora hinnulea, Sporotrichum pruinosum, Thielavia cf.microspora, Aspergillus sp., Scopulariopsis sp., Fusarium sp.,Verticillium sp., Pseudoplectania nigrella, and Phytophthora infestans.

Preferably, the polypeptides of the present invention comprise the aminoacid sequence of the polypeptide encoded by the cellobiohydrolase Iencoding part of the nucleotide sequence inserted into a plasmid presentin a deposited microorganism selected from the group consisting of CGMCCNo. 0584, CGMCC No. 0581, CGMCC No. 0585, CGMCC No. 0582, CGMCC No.0583, CBS109513, DSM 14348, CGMCC No. 0580, DSM 15064, DSM 15065, DSM15066, DSM 15067, CGMCC No. 0747, CGMCC No. 0748, CGMCC No. 0749, andCGMCC No. 0750. In another preferred embodiment, the polypeptide of thepresent invention consists of the amino acid sequence of the polypeptideencoded by the cellobiohydrolase I encoding part of the nucleotidesequence inserted into a plasmid present in a deposited microorganismselected from the group consisting of CGMCC No. 0584, CGMCC No. 0581,CGMCC No. 0585, CGMCC No. 0582, CGMCC No. 0583, CBS109513, DSM 14348,and CGMCC No. 0580, DSM 15064, DSM 15065, DSM 15066, DSM 15067, CGMCCNo. 0747, CGMCC No. 0748, CGMCC No. 0749, and CGMCC No. 0750.

In a similar way as described above, the polypeptide of the inventionmay be an artificial variant which comprises, preferably consists of, anamino acid sequence that has at least one substitution, deletion and/orinsertion of an amino acid as compared to the amino acid sequenceencoded by the cellobiohydrolase I encoding part of the nucleotidesequence inserted into a plasmid present in a deposited microorganismselected from the group consisting of CGMCC No. 0584, CGMCC No. 0581,CGMCC No. 0585, CGMCC No. 0582, CGMCC No. 0583, CBS109513, DSM 14348,and CGMCC No. 0580, DSM 15064, DSM 15065, DSM 15066, DSM 15067, CGMCCNo. 0747, CGMCC No. 0748, CGMCC No. 0749, and CGMCC No. 0750.

In a third embodiment, the present invention relates to polypeptideshaving cellobiohydrolase I activity which are encoded by nucleotidesequences which hybridize under very low stringency conditions,preferably under low stringency conditions, more preferably under mediumstringency conditions, more preferably under medium-high stringencyconditions, even more preferably under high stringency conditions, andmost preferably under very high stringency conditions with apolynucleotide probe selected from the group consisting of

(i) the complementary strand of the nucleotides selected from the groupconsisting of:

nucleotides 1 to 1578 of SEQ ID NO:1,

nucleotides 1 to 1587 of SEQ ID NO:3,

nucleotides 1 to 1353 of SEQ ID NO:5,

nucleotides 1 to 1371 of SEQ ID NO:7,

nucleotides 1 to 1614 of SEQ ID NO:9,

nucleotides 1 to 1245 of SEQ ID NO:11,

nucleotides 1 to 1341 of SEQ ID NO:13,

nucleotides 1 to 1356 of SEQ ID NO:15,

nucleotides 1 to 1365 of SEQ ID NO:37,

nucleotides 1 to 1377 of SEQ ID NO:39,

nucleotides 1 to 1353 of SEQ ID NO:41,

nucleotides 1 to 1341 of SEQ ID NO:43,

nucleotides 1 to 1584 of SEQ ID NO:45,

nucleotides 1 to 1368 of SEQ ID NO:47,

nucleotides 1 to 1395 of SEQ ID NO:49,

nucleotides 1 to 1383 of SEQ ID NO:51,

nucleotides 1 to 1353 of SEQ ID NO:53,

nucleotides 1 to 1599 of SEQ ID NO:55,

nucleotides 1 to 1383 of SEQ ID NO:57,

nucleotides 1 to 1578 of SEQ ID NO:59, and

nucleotides 1 to 1371 of SEQ ID NO:65;

(ii) the complementary strand of the nucleotides selected from the groupconsisting of

nucleotides 1 to 500 of SEQ ID NO:1,

nucleotides 1 to 500 of SEQ ID NO:3,

nucleotides 1 to 500 of SEQ ID NO:5,

nucleotides 1 to 500 of SEQ ID NO:7,

nucleotides 1 to 500 of SEQ ID NO:9,

nucleotides 1 to 500 of SEQ ID NO:11,

nucleotides 1 to 500 of SEQ ID NO:13,

nucleotides 1 to 500 of SEQ ID NO:15,

nucleotides 1 to 500 of SEQ ID NO:37,

nucleotides 1 to 500 of SEQ ID NO:39,

nucleotides 1 to 500 of SEQ ID NO:41,

nucleotides 1 to 500 of SEQ ID NO:43,

nucleotides 1 to 500 of SEQ ID NO:45,

nucleotides 1 to 500 of SEQ ID NO:47,

nucleotides 1 to 500 of SEQ ID NO:49,

nucleotides 1 to 500 of SEQ ID NO:51,

nucleotides 1 to 500 of SEQ ID NO:53,

nucleotides 1 to 500 of SEQ ID NO:55,

nucleotides 1 to 500 of SEQ ID NO:57,

nucleotides 1 to 500 of SEQ ID NO:59,

nucleotides 1 to 500 of SEQ ID NO:65,

nucleotides 1 to 221 of SEQ ID NO:17,

nucleotides 1 to 239 of SEQ ID NO:18,

nucleotides 1 to 199 of SEQ ID NO:19,

nucleotides 1 to 191 of SEQ ID NO:20,

nucleotides 1 to 232 of SEQ ID NO:21,

nucleotides 1 to 467 of SEQ ID NO:22,

nucleotides 1 to 534 of SEQ ID NO:23,

nucleotides 1 to 563 of SEQ ID NO:24,

nucleotides 1 to 218 of SEQ ID NO:25,

nucleotides 1 to 492 of SEQ ID NO:26,

nucleotides 1 to 481 of SEQ ID NO:27,

nucleotides 1 to 463 of SEQ ID NO:28,

nucleotides 1 to 513 of SEQ ID NO:29,

nucleotides 1 to 579 of SEQ ID NO:30,

nucleotides 1 to 514 of SEQ ID NO:31,

nucleotides 1 to 477 of SEQ ID NO:32,

nucleotides 1 to 500 of SEQ ID NO:33,

nucleotides 1 to 470 of SEQ ID NO:34,

nucleotides 1 to 491 of SEQ ID NO:35,

nucleotides 1 to 221 of SEQ ID NO:36,

nucleotides 1 to 519 of SEQ ID NO:61,

nucleotides 1 to 497 of SEQ ID NO:62,

nucleotides 1 to 498 of SEQ ID NO:63,

nucleotides 1 to 525 of SEQ ID NO:64, and

nucleotides 1 to 951 of SEQ ID NO:67; and

(iii) the complementary strand of the nucleotides selected from thegroup consisting of

nucleotides 1 to 200 of SEQ ID NO:1,

nucleotides 1 to 200 of SEQ ID NO:3,

nucleotides 1 to 200 of SEQ ID NO:5,

nucleotides 1 to 200 of SEQ ID NO:7,

nucleotides 1 to 200 of SEQ ID NO:9,

nucleotides 1 to 200 of SEQ ID NO:11,

nucleotides 1 to 200 of SEQ ID NO:13,

nucleotides 1 to 200 of SEQ ID NO:15,

nucleotides 1 to 200 of SEQ ID NO:37,

nucleotides 1 to 200 of SEQ ID NO:39,

nucleotides 1 to 200 of SEQ ID NO:41,

nucleotides 1 to 200 of SEQ ID NO:43,

nucleotides 1 to 200 of SEQ ID NO:45,

nucleotides 1 to 200 of SEQ ID NO:47,

nucleotides 1 to 200 of SEQ ID NO:49,

nucleotides 1 to 200 of SEQ ID NO:51,

nucleotides 1 to 200 of SEQ ID NO:53,

nucleotides 1 to 200 of SEQ ID NO:55,

nucleotides 1 to 200 of SEQ ID NO:57,

nucleotides 1 to 200 of SEQ ID NO:59, and

nucleotides 1 to 200 of SEQ ID NO:65

(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.).

In another embodiment, the present invention relates to polypeptideshaving cellobiohydrolase I activity which are encoded by thecellobiohydrolase I encoding part of the nucleotide sequence present ina microorganism selected from the group consisting of:

a microorganism belonging to Zygomycota, preferably belonging to theMucorales, more preferably belonging to the family Mucoraceae, mostpreferably belonging to the genus Rhizomucor (e.g., Rhizomucorpusillus), or the family Choanephoraceae, most preferably belonging tothe genus Poitrasia (e.g., Poitrasia circinans),

a microorganism belonging to the Oomycetes, preferably to the orderPythiales, more preferably to the family Pythiaceae, most preferably tothe genus Phytophthora (e.g., Phytophthora infestans),

a microorganism belonging to Auriculariales (an order of theBasidiomycota, Hymenomycetes), preferably belonging to the familyExidiaceae, more preferably belonging to the genus Exidia (e.g., Exidiaglandulosa),

a microorganism belonging to Xylariales (an order of the Ascomycota,Sordariomycetes), preferably belonging to the family Xylariaceae, morepreferably belonging to the genus Xylaria (e.g., Xylaria hypoxylon),

a microorganism belonging to Dothideales (an order of the Ascomycota,Dothideomycetes), preferably belonging to the family Dothideaceae, morepreferably belonging to the genus Diplodia (e.g., Diplodia gossypina),

a microorganism belonging to Pezizales (an order of the Ascomycota),preferably belonging to the family Pyronemataceae, more preferablybelonging to the genus Trichophaea (e.g., Trichophaea saccata), or thefamily Sarcosomataceae, more preferably belonging to the genusPseudoplectania (e.g., Pseudoplectania nigrella),

a microorganism belonging to the family Rigidiporaceae (underBasidiomycota, Hymenomycetes, Hymenomycetales), more preferablybelonging to the genus Meripilus (e.g., Meripilus giganteus),

a microorganism belonging to the family Meruliaceae (underBasidiomycota, Hymenomycetes, Sterealesales), more preferably belongingto the genus Sporothrichum (Sporothrichum sp.),

a microorganism belonging to the family Agaricaceae (underBasidiomycota, Hymenomycetes, Agaricales), more preferably belonging tothe genus Coprinus (e.g., Coprinus cinereus),

a microorganism belonging to the family Hypocreaceae (under Ascomycota,Sordariomycetes, Hypocreales), more preferably belonging to the genusAcremonium (e.g., Acremonium thermophilum; Acremonium sp.) or the(mitosporic) genus Verticillium (e.g., Verticillium tenerum),

a microorganism belonging to the genus Cladorrhinum (under Ascomycota,Sordariomycetes, Sordariales, Sordariaceae) e.g., Cladorrhinumfoecundissimum,

a microorganism belonging to the genus Myceliophthora (under Ascomycota,Sordariomycetes, Sordariales, Sordariaceae) e.g., Myceliophthorathermophila or Myceliophthora hinnulae,

a microorganism belonging to the genus Chaetomium (under Ascomycota,Sordariomycetes, Sordariales, Chaetomiaceae) e.g., Chaetomiumthermophilum,

a microorganism belonging to the genus Chaetomidium (under Ascomycota,Sordariomycetes, Sordariales, Chaetomiaceae) e.g., Chaetomidiumpingtungium,

a microorganism belonging to the genus Thielavia (under Ascomycota,Sordariomycetes, Sordariales, Chaetomiaceae) e.g., Thielaviaaustraliensis or Thielavia microspora,

a microorganism belonging to the genus Thermoascus (under Ascomycota,Eurotiomycetes, Eurotiales, Trichocomoaceae) e.g., Thermoascusaurantiacus,

a microorganism belonging to the genus Trichothecium (mitosporicAscomycota) e.g., Trichothecium roseum, and

a microorganism belonging to the species Humicola nigrescens.

A nucleotide sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:65, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:67, or asubsequence thereof, as well as an amino acid sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38,SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,SEQ ID NO:60, and SEQ ID NO:66, or a fragment thereof, may be used todesign a polynucleotide probe to identify and clone DNA encodingpolypeptides having cellobiohydrolase I activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,more preferably at least 35 nucleotides in length, such as at least 70nucleotides in length. It is, however, preferred that the polynucleotideprobe is at least 100 nucleotides in length. For example, thepolynucleotide probe may be at least 200 nucleotides in length, at least300 nucleotides in length, at least 400 nucleotides in length or atleast 500 nucleotides in length. Even longer probes may be used, e.g.,polynucleotide probes which are at least 600 nucleotides in length, atleast 700 nucleotides in length, at least 800 nucleotides in length, orat least 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin).

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand which encodes a polypeptide having cellobiohydrolase I activity.Genomic or other DNA from such other organisms may be separated byagarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to, and immobilized, on nitrocellulose or other suitablecarrier materials. In order to identify a clone or DNA which ishomologous with SEQ ID NO:1 the carrier material with the immobilizedDNA is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled polynucleotide probe whichhybridizes to the nucleotide sequence shown in SEQ ID NO:1 under verylow to very high stringency conditions. Molecules to which thepolynucleotide probe hybridizes under these conditions may be detectedusing X-ray film or by any other method known in the art. Whenever theterm “polynucleotide probe” is used in the present context, it is to beunderstood that such a probe contains at least 15 nucleotides.

In an interesting embodiment, the polynucleotide probe is thecomplementary strand of the nucleotides selected from the groupconsisting of:

nucleotides 1 to 1578 of SEQ ID NO:1,

nucleotides 1 to 1302 of SEQ ID NO:1,

nucleotides 1 to 1587 of SEQ ID NO:3,

nucleotides 1 to 1302 of SEQ ID NO:3,

nucleotides 1 to 1353 of SEQ ID NO:5,

nucleotides 1 to 1302 of SEQ ID NO:5,

nucleotides 1 to 1371 of SEQ ID NO:7,

nucleotides 1 to 1302 of SEQ ID NO:7,

nucleotides 1 to 1614 of SEQ ID NO:9,

nucleotides 1 to 1302 of SEQ ID NO:9,

nucleotides 1 to 1245 of SEQ ID NO:11,

nucleotides 1 to 1341 of SEQ ID NO:13,

nucleotides 1 to 1302 of SEQ ID NO:13,

nucleotides 1 to 1356 of SEQ ID NO:15,

nucleotides 1 to 1302 of SEQ ID NO:15,

nucleotides 1 to 1365 of SEQ ID NO:37,

nucleotides 1 to 1302 of SEQ ID NO:37,

nucleotides 1 to 1377 of SEQ ID NO:39,

nucleotides 1 to 1302 of SEQ ID NO:39,

nucleotides 1 to 1353 of SEQ ID NO:41,

nucleotides 1 to 1302 of SEQ ID NO:41,

nucleotides 1 to 1341 of SEQ ID NO:43,

nucleotides 1 to 1302 of SEQ ID NO:43,

nucleotides 1 to 1584 of SEQ ID NO:45,

nucleotides 1 to 1302 of SEQ ID NO:45,

nucleotides 1 to 1368 of SEQ ID NO:47,

nucleotides 1 to 1302 of SEQ ID NO:47,

nucleotides 1 to 1395 of SEQ ID NO:49,

nucleotides 1 to 1302 of SEQ ID NO:49,

nucleotides 1 to 1383 of SEQ ID NO:51,

nucleotides 1 to 1302 of SEQ ID NO:51,

nucleotides 1 to 1353 of SEQ ID NO:53,

nucleotides 1 to 1302 of SEQ ID NO:53,

nucleotides 1 to 1599 of SEQ ID NO:55,

nucleotides 1 to 1302 of SEQ ID NO:55,

nucleotides 1 to 1383 of SEQ ID NO:57,

nucleotides 1 to 1302 of SEQ ID NO:57,

nucleotides 1 to 1578 of SEQ ID NO:59,

nucleotides 1 to 1302 of SEQ ID NO:59,

nucleotides 1 to 1371 of SEQ ID NO:65, and

nucleotides 1 to 1302 of SEQ ID NO:65;

or the complementary strand of the nucleotides selected from the groupconsisting of:

nucleotides 1 to 500 of SEQ ID NO:1,

nucleotides 1 to 500 of SEQ ID NO:3,

nucleotides 1 to 500 of SEQ ID NO:5,

nucleotides 1 to 500 of SEQ ID NO:7,

nucleotides 1 to 500 of SEQ ID NO:9,

nucleotides 1 to 500 of SEQ ID NO:11,

nucleotides 1 to 500 of SEQ ID NO:13,

nucleotides 1 to 500 of SEQ ID NO:15,

nucleotides 1 to 500 of SEQ ID NO:37,

nucleotides 1 to 500 of SEQ ID NO:39,

nucleotides 1 to 500 of SEQ ID NO:41,

nucleotides 1 to 500 of SEQ ID NO:43,

nucleotides 1 to 500 of SEQ ID NO:45,

nucleotides 1 to 500 of SEQ ID NO:47,

nucleotides 1 to 500 of SEQ ID NO:49,

nucleotides 1 to 500 of SEQ ID NO:51,

nucleotides 1 to 500 of SEQ ID NO:53,

nucleotides 1 to 500 of SEQ ID NO:55,

nucleotides 1 to 500 of SEQ ID NO:57,

nucleotides 1 to 500 of SEQ ID NO:59,

nucleotides 1 to 500 of SEQ ID NO:65,

nucleotides 1 to 221 of SEQ ID NO:17,

nucleotides 1 to 239 of SEQ ID NO:18,

nucleotides 1 to 199 of SEQ ID NO:19,

nucleotides 1 to 191 of SEQ ID NO:20,

nucleotides 1 to 232 of SEQ ID NO:21,

nucleotides 1 to 467 of SEQ ID NO:22,

nucleotides 1 to 534 of SEQ ID NO:23,

nucleotides 1 to 563 of SEQ ID NO:24,

nucleotides 1 to 218 of SEQ ID NO:25,

nucleotides 1 to 492 of SEQ ID NO:26,

nucleotides 1 to 481 of SEQ ID NO:27,

nucleotides 1 to 463 of SEQ ID NO:28,

nucleotides 1 to 513 of SEQ ID NO:29,

nucleotides 1 to 579 of SEQ ID NO:30,

nucleotides 1 to 514 of SEQ ID NO:31,

nucleotides 1 to 477 of SEQ ID NO:32,

nucleotides 1 to 500 of SEQ ID NO:33,

nucleotides 1 to 470 of SEQ ID NO:34,

nucleotides 1 to 491 of SEQ ID NO:35,

nucleotides 1 to 221 of SEQ ID NO:36,

nucleotides 1 to 519 of SEQ ID NO:61,

nucleotides 1 to 497 of SEQ ID NO:62,

nucleotides 1 to 498 of SEQ ID NO:63,

nucleotides 1 to 525 of SEQ ID NO:64, and

nucleotides 1 to 951 of SEQ ID NO:67;

or the complementary strand of the nucleotides selected from the groupconsisting of:

nucleotides 1 to 200 of SEQ ID NO:1,

nucleotides 1 to 200 of SEQ ID NO:3,

nucleotides 1 to 200 of SEQ ID NO:5,

nucleotides 1 to 200 of SEQ ID NO:7,

nucleotides 1 to 200 of SEQ ID NO:9,

nucleotides 1 to 200 of SEQ ID NO:11,

nucleotides 1 to 200 of SEQ ID NO:13,

nucleotides 1 to 200 of SEQ ID NO:15,

nucleotides 1 to 200 of SEQ ID NO:37,

nucleotides 1 to 200 of SEQ ID NO:39,

nucleotides 1 to 200 of SEQ ID NO:41,

nucleotides 1 to 200 of SEQ ID NO:43,

nucleotides 1 to 200 of SEQ ID NO:45,

nucleotides 1 to 200 of SEQ ID NO:47,

nucleotides 1 to 200 of SEQ ID NO:49,

nucleotides 1 to 200 of SEQ ID NO:51,

nucleotides 1 to 200 of SEQ ID NO:53,

nucleotides 1 to 200 of SEQ ID NO:55,

nucleotides 1 to 200 of SEQ ID NO:57,

nucleotides 1 to 200 of SEQ ID NO:59,

nucleotides 1 to 200 of SEQ ID NO:65,

nucleotides 1 to 200 of SEQ ID NO:22,

nucleotides 1 to 200 of SEQ ID NO:23,

nucleotides 1 to 200 of SEQ ID NO:24,

nucleotides 1 to 200 of SEQ ID NO:26,

nucleotides 1 to 200 of SEQ ID NO:27,

nucleotides 1 to 200 of SEQ ID NO:28,

nucleotides 1 to 200 of SEQ ID NO:29,

nucleotides 1 to 200 of SEQ ID NO:30,

nucleotides 1 to 200 of SEQ ID NO:31,

nucleotides 1 to 200 of SEQ ID NO:32,

nucleotides 1 to 200 of SEQ ID NO:33,

nucleotides 1 to 200 of SEQ ID NO:34,

nucleotides 1 to 200 of SEQ ID NO:35,

nucleotides 1 to 200 of SEQ ID NO:61,

nucleotides 1 to 200 of SEQ ID NO:62,

nucleotides 1 to 200 of SEQ ID NO:63,

nucleotides 1 to 200 of SEQ ID NO:64, and

nucleotides 1 to 200 of SEQ ID NO:67.

In another interesting embodiment, the polynucleotide probe is thecomplementary strand of the nucleotide sequence which encodes apolypeptide selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:66. In afurther interesting embodiment, the polynucleotide probe is thecomplementary strand of a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, and SEQ ID NO:65. In another interesting embodiment, thepolynucleotide probe is the complementary strand of the nucleotidesequence contained in a plasmid which is contained in a depositedmicroorganism selected from the group consisting of CGMCC No. 0584,CGMCC No. 0581, CGMCC No. 0585, CGMCC No. 0582, CGMCC No. 0583, CGMCCNo. 0580, CBS 109513, DSM 14348, DSM 15064, DSM 15065, DSM 15066, DSM15067, CGMCC No. 0747, CGMCC No. 0748, CGMCC No. 0749, and CGMCC No.0750.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 1.0% SDS, 5×Denhardt's solution, 100micrograms/ml sheared and denatured salmon sperm DNA, following standardSouthern blotting procedures. Preferably, the long probes of at least100 nucleotides do not contain more than 1000 nucleotides. For longprobes of at least 100 nucleotides in length, the carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.1% SDS at42° C. (very low stringency), preferably washed three times each for 15minutes using 0.5×SSC, 0.1% SDS at 42° C. (low stringency), morepreferably washed three times each for 15 minutes using 0.2×SSC, 0.1%SDS at 42° C. (medium stringency), even more preferably washed threetimes each for 15 minutes using 0.2×SSC, 0.1% SDS at 55° C. (medium-highstringency), most preferably washed three times each for 15 minutesusing 0.1×SSC, 0.1% SDS at 60° C. (high stringency), in particularwashed three times each for 15 minutes using 0.1×SSC, 0.1% SDS at 68° C.(very high stringency).

Although not particularly preferred, it is contemplated that shorterprobes, e.g., probes which are from about 15 to 99 nucleotides inlength, such as from about 15 to about 70 nucleotides in length, may bealso be used. For such short probes, stringency conditions are definedas prehybridization, hybridization, and washing post-hybridization at 5°C. to 10° C. below the calculated T_(m) using the calculation accordingto Bolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to 99 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SDS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

Sources for Polypeptides Having Cellobiohydrolase I Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein shall mean that the polypeptideencoded by the nucleotide sequence is produced by a cell in which thenucleotide sequence is naturally present or into which the nucleotidesequence has been inserted. In a preferred embodiment, the polypeptideis secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide; or a Streptomyces polypeptide, e.g., a Streptomyceslividans or Streptomyces murinus polypeptide; or a gram negativebacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

A polypeptide of the present invention may be a fungal polypeptide, andmore preferably a yeast polypeptide such as a Candida, Kluyveromyces,Neocallimastix, Pichia, Piromyces, Saccharomyces, Schizosaccharomyces,or Yarrowia polypeptide; or more preferably a filamentous fungalpolypeptide such as an Acremonium, Aspergillus, Aureobasidium,Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,Myceliophthora, Neurospora, Paecilomyces, Penicillium, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide.

In an interesting embodiment, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis polypeptide.

In another interesting embodiment, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide.

In a preferred embodiment, the polypeptide is a Acremonium thermophilum,Chaetomium thermophilum, Scytalidium sp., Scytalidium thermophilum,Thermoascus aurantiacus, Thielavia australiensis, Verticillium tenerum,Neotermes castaneus, Melanocarpus albomyces, Poitrasia circinans,Coprinus cinereus, Trichothecium roseum, Humicola nigrescens,Cladorrhinum foecundissimum, Diplodia gossypina, Myceliophthorathermophila, Rhizomucor pusillus, Meripilus giganteus, Exidiaglandulosa, Xylaria hypoxylon, Trichophaea saccata, Acremonium sp.,Chaetomium sp., Chaetomidium pingtungium, Myceliophthora thermophila,Myceliophthora hinnulea, Sporotrichum pruinosum, Thielavia cf.microspora, Aspergillus sp., Scopulariopsis sp., Fusarium sp.,Verticillium sp., Pseudoplectania nigrella, or Phytophthora infestanspolypeptide.

In a more preferred embodiment, the polypeptide is a Acremoniumthermophilum, Chaetomium thermophilum, Scytalidium sp., Scytalidiumthermophilum, Thermoascus aurantiacus, Thielavia australiensis,Verticillium tenerum, Neotermes castaneus, Melanocarpus albomyces,Poitrasia circinans, or Coprinus cinereus polypeptide, e.g., thepolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, and SEQ ID NO:66.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), China General Microbiological CultureCollection Center (CGMCC), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,water, plants, animals, etc.) using the above-mentioned probes.Techniques for isolating microorganisms from natural habitats are wellknown in the art. The nucleotide sequence may then be derived bysimilarly screening a genomic or cDNA library of another microorganism.Once a nucleotide sequence encoding a polypeptide has been detected withthe probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polypeptides encoded by nucleotide sequences of the present inventionalso include fused polypeptides or cleavable fusion polypeptides inwhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleotide sequence (or a portion thereof) encoding anotherpolypeptide to a nucleotide sequence (or a portion thereof) of thepresent invention. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

Polynucleotides and Nucleotide Sequences

The present invention also relates to polynucleotides having anucleotide sequence which encodes for a polypeptide of the invention. Inparticular, the present invention relates to polynucleotides consistingof a nucleotide sequence which encodes for a polypeptide of theinvention. In a preferred embodiment, the nucleotide sequence isselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, and SEQ ID NO:65. In a more preferredembodiment, the nucleotide sequence is the mature polypeptide codingregion contained in a plasmid which is contained in a depositedmicroorganism selected from the group consisting of CGMCC No. 0584,CGMCC No. 0581, CGMCC No. 0585, CGMCC No. 0582, CGMCC No. 0583, CGMCCNo. 0580, CBS109513, DSM 14348, DSM 15064, DSM 15065, DSM 15066, DSM15067, CGMCC No. 0747, CGMCC No. 0748, CGMCC No. 0749, and CGMCC No.0750. The present invention also encompasses polynucleotides comprising,preferably consisting of, nucleotide sequences which encode apolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, and SEQ ID NO:66, which differ from a nucleotide sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, and SEQ ID NO:65 by virtue of thedegeneracy of the genetic code.

The present invention also relates to polynucleotides comprising,preferably consisting of, a subsequence of a nucleotide sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, and SEQ ID NO:65 which encodefragments of an amino acid sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQID NO:66 that have cellobiohydrolase I activity. A subsequence of anucleotide sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, and SEQ ID NO:65 is anucleotide sequence encompassed by a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, and SEQ ID NO:65 except that one or more nucleotides from the 5′and/or 3′ end have been deleted.

The present invention also relates to polynucleotides having, preferablyconsisting of, a modified nucleotide sequence which comprises at leastone modification in the mature polypeptide coding sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, and SEQ ID NO:65, and where the modified nucleotidesequence encodes a polypeptide which consists of an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:66.

The techniques used to isolate or clone a nucleotide sequence encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thenucleotide sequences of the present invention from such genomic DNA canbe effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other amplification procedures such as ligase chain reaction (LCR),ligated activated transcription (LAT) and nucleotide sequence-basedamplification (NASBA) may be used. The nucleotide sequence may be clonedfrom a strain selected from the group consisting of Acremonium,Scytalidium, Thermoascus, Thielavia, Verticillium, Neotermes,Melanocarpus, Poitrasia, Coprinus, Trichothecium, Humicola,Cladorrhinum, Diplodia, Myceliophthora, Rhizomucor, Meripilus, Exidia,Xylaria, Trichophaea, Chaetomium, Chaetomidium, Sporotrichum, Thielavia,Aspergillus, Scopulariopsis, Fusarium, Pseudoplectania, andPhytophthora, or another or related organism and thus, for example, maybe an allelic or species variant of the polypeptide encoding region ofthe nucleotide sequence.

The nucleotide sequence may be obtained by standard cloning proceduresused in genetic engineering to relocate the nucleotide sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desiredfragment comprising the nucleotide sequence encoding the polypeptide,insertion of the fragment into a vector molecule, and incorporation ofthe recombinant vector into a host cell where multiple copies or clonesof the nucleotide sequence will be replicated. The nucleotide sequencemay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

The present invention also relates to a polynucleotide comprising,preferably consisting of, a nucleotide sequence which has a degree ofidentity with a nucleotide sequence selected from the group consistingof

nucleotides 1 to 1578 of SEQ ID NO:1,

nucleotides 1 to 1587 of SEQ ID NO:3,

nucleotides 1 to 1353 of SEQ ID NO:5,

nucleotides 1 to 1371 of SEQ ID NO:7,

nucleotides 1 to 1614 of SEQ ID NO:9,

nucleotides 1 to 1245 of SEQ ID NO:11,

nucleotides 1 to 1341 of SEQ ID NO:13,

nucleotides 1 to 1356 of SEQ ID NO:15,

nucleotides 1 to 1365 of SEQ ID NO:37,

nucleotides 1 to 1377 of SEQ ID NO:39,

nucleotides 1 to 1353 of SEQ ID NO:41,

nucleotides 1 to 1341 of SEQ ID NO:43,

nucleotides 1 to 1584 of SEQ ID NO:45,

nucleotides 1 to 1368 of SEQ ID NO:47,

nucleotides 1 to 1395 of SEQ ID NO:49,

nucleotides 1 to 1383 of SEQ ID NO:51,

nucleotides 1 to 1353 of SEQ ID NO:53,

nucleotides 1 to 1599 of SEQ ID NO:55,

nucleotides 1 to 1383 of SEQ ID NO:57,

nucleotides 1 to 1578 of SEQ ID NO:59,

nucleotides 1 to 1371 of SEQ ID NO:65,

nucleotides 1 to 500 of SEQ ID NO:1,

nucleotides 1 to 500 of SEQ ID NO:3,

nucleotides 1 to 500 of SEQ ID NO:5,

nucleotides 1 to 500 of SEQ ID NO:7,

nucleotides 1 to 500 of SEQ ID NO:9,

nucleotides 1 to 500 of SEQ ID NO:11,

nucleotides 1 to 500 of SEQ ID NO:13,

nucleotides 1 to 500 of SEQ ID NO:15,

nucleotides 1 to 500 of SEQ ID NO:37,

nucleotides 1 to 500 of SEQ ID NO:39,

nucleotides 1 to 500 of SEQ ID NO:41,

nucleotides 1 to 500 of SEQ ID NO:43,

nucleotides 1 to 500 of SEQ ID NO:45,

nucleotides 1 to 500 of SEQ ID NO:47,

nucleotides 1 to 500 of SEQ ID NO:49,

nucleotides 1 to 500 of SEQ ID NO:51,

nucleotides 1 to 500 of SEQ ID NO:53,

nucleotides 1 to 500 of SEQ ID NO:55,

nucleotides 1 to 500 of SEQ ID NO:57,

nucleotides 1 to 500 of SEQ ID NO:59,

nucleotides 1 to 500 of SEQ ID NO:65,

nucleotides 1 to 221 of SEQ ID NO:17,

nucleotides 1 to 239 of SEQ ID NO:18,

nucleotides 1 to 199 of SEQ ID NO:19,

nucleotides 1 to 191 of SEQ ID NO:20,

nucleotides 1 to 232 of SEQ ID NO:21,

nucleotides 1 to 467 of SEQ ID NO:22,

nucleotides 1 to 534 of SEQ ID NO:23,

nucleotides 1 to 563 of SEQ ID NO:24,

nucleotides 1 to 218 of SEQ ID NO:25,

nucleotides 1 to 492 of SEQ ID NO:26,

nucleotides 1 to 481 of SEQ ID NO:27,

nucleotides 1 to 463 of SEQ ID NO:28,

nucleotides 1 to 513 of SEQ ID NO:29,

nucleotides 1 to 579 of SEQ ID NO:30,

nucleotides 1 to 514 of SEQ ID NO:31,

nucleotides 1 to 477 of SEQ ID NO:32,

nucleotides 1 to 500 of SEQ ID NO:33,

nucleotides 1 to 470 of SEQ ID NO:34,

nucleotides 1 to 491 of SEQ ID NO:35,

nucleotides 1 to 221 of SEQ ID NO:36,

nucleotides 1 to 519 of SEQ ID NO:61,

nucleotides 1 to 497 of SEQ ID NO:62,

nucleotides 1 to 498 of SEQ ID NO:63,

nucleotides 1 to 525 of SEQ ID NO:64, and

nucleotides 1 to 951 of SEQ ID NO:67

of at least 70% identity, such as at least 75% identity; preferably, thenucleotide sequence has at least 80% identity, e.g., at least 85%identity, such as at least 90% identity, more preferably at least 95%identity, such as at least 96% identity, e.g., at least 97% identity,even more preferably at least 98% identity, such as at least 99%.Preferably, the nucleotide sequence encodes a polypeptide havingcellobiohydrolase I activity. The degree of identity between twonucleotide sequences is determined as described previously (see thesection entitled “Definitions”).

In another interesting aspect, the present invention relates to apolynucleotide having, preferably consisting of, a nucleotide sequencewhich has at least 65% identity with the cellobiohydrolase I encodingpart of the nucleotide sequence inserted into a plasmid present in adeposited microorganism selected from the group consisting of CGMCC No.0584, CGMCC No. 0581, CGMCC No. 0585, CGMCC No. 0582, CGMCC No. 0583,CGMCC No. 0580, CBS 109513, DSM 14348, DSM 15064, DSM 15065, DSM 15066,DSM 15067, CGMCC No. 0747, CGMCC No. 0748, CGMCC No. 0749, and CGMCC No.0750. In a preferred embodiment, the degree of identity with thecellobiohydrolase I encoding part of the nucleotide sequence insertedinto a plasmid present in a deposited microorganism selected from thegroup consisting of CGMCC No. 0584, CGMCC No. 0581, CGMCC No. 0585,CGMCC No. 0582, CGMCC No. 0583, CGMCC No. 0580, CBS109513, DSM 14348,DSM 15064, DSM 15065, DSM 15066, DSM 15067, CGMCC No. 0747, CGMCC No.0748, CGMCC No. 0749, and CGMCC No. 0750 is at least 70%, e.g., at least80%, such as at least 90%, more preferably at least 95%, such as atleast 96%, e.g., at least 97%, even more preferably at least 98%, suchas at least 99%. Preferably, the nucleotide sequence comprises thecellobiohydrolase I encoding part of the nucleotide sequence insertedinto a plasmid present in a deposited microorganism selected from thegroup consisting of CGMCC No. 0584, CGMCC No. 0581, CGMCC No. 0585,CGMCC No. 0582, CGMCC No. 0583, CGMCC No. 0580, CBS109513, DSM 14348,DSM 15064, DSM 15065, DSM 15066, DSM 15067, CGMCC No. 0747, CGMCC No.0748, CGMCC No. 0749, and CGMCC No. 0750. In an even more preferredembodiment, the nucleotide sequence consists of the cellobiohydrolase Iencoding part of the nucleotide sequence inserted into a plasmid presentin a deposited microorganism selected from the group consisting of CGMCCNo. 0584, CGMCC No. 0581, CGMCC No. 0585, CGMCC No. 0582, CGMCC No.0583, CGMCC No. 0580, CBS109513, DSM 14348, DSM 15064, DSM 15065, DSM15066, DSM 15067, CGMCC No. 0747, CGMCC No. 0748, CGMCC No. 0749, andCGMCC No. 0750.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of a polypeptide,which comprises an amino acid sequence that has at least onesubstitution, deletion and/or insertion as compared to an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:66. These artificialvariants may differ in some engineered way from the polypeptide isolatedfrom its native source, e.g., variants that differ in specific activity,thermostability, pH optimum, or the like.

It will be apparent to those skilled in the art that such modificationscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the nucleotide sequence ofthe invention, and therefore preferably not subject to modification,such as substitution, may be identified according to procedures known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, mutations are introduced at everypositively charged residue in the molecule, and the resultant mutantmolecules are tested for cellobiohydrolase I activity to identify aminoacid residues that are critical to the activity of the molecule. Sitesof substrate-enzyme interaction can also be determined by analysis ofthe three-dimensional structure as determined by such techniques asnuclear magnetic resonance analysis, crystallography or photoaffinitylabelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smithet al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver etal., 1992, FEBS Letters 309: 59-64).

Moreover, a nucleotide sequence encoding a polypeptide of the presentinvention may be modified by introduction of nucleotide substitutionswhich do not give rise to another amino acid sequence of the polypeptideencoded by the nucleotide sequence, but which correspond to the codonusage of the host organism intended for production of the enzyme.

The introduction of a mutation into the nucleotide sequence to exchangeone nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure, which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with Dpnl which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

The present invention also relates to a polynucleotide comprising,preferably consisting of, a nucleotide sequence which encodes apolypeptide having cellobiohydrolase I activity, and which hybridizesunder very low stringency conditions, preferably under low stringencyconditions, more preferably under medium stringency conditions, morepreferably under medium-high stringency conditions, even more preferablyunder high stringency conditions, and most preferably under very highstringency conditions with a polynucleotide probe selected from thegroup consisting of

(i) the complementary strand of the nucleotides selected from the groupconsisting of:

nucleotides 1 to 1578 of SEQ ID NO:1,

nucleotides 1 to 1302 of SEQ ID NO:1,

nucleotides 1 to 1587 of SEQ ID NO:3,

nucleotides 1 to 1302 of SEQ ID NO:3,

nucleotides 1 to 1353 of SEQ ID NO:5,

nucleotides 1 to 1302 of SEQ ID NO:5,

nucleotides 1 to 1371 of SEQ ID NO:7,

nucleotides 1 to 1302 of SEQ ID NO:7,

nucleotides 1 to 1614 of SEQ ID NO:9,

nucleotides 1 to 1302 of SEQ ID NO:9,

nucleotides 1 to 1245 of SEQ ID NO:11,

nucleotides 1 to 1341 of SEQ ID NO:13,

nucleotides 1 to 1302 of SEQ ID NO:13,

nucleotides 1 to 1356 of SEQ ID NO:15,

nucleotides 1 to 1302 of SEQ ID NO:15,

nucleotides 1 to 1365 of SEQ ID NO:37,

nucleotides 1 to 1302 of SEQ ID NO:37,

nucleotides 1 to 1377 of SEQ ID NO:39,

nucleotides 1 to 1302 of SEQ ID NO:39,

nucleotides 1 to 1353 of SEQ ID NO:41,

nucleotides 1 to 1302 of SEQ ID NO:41,

nucleotides 1 to 1341 of SEQ ID NO:43,

nucleotides 1 to 1302 of SEQ ID NO:43,

nucleotides 1 to 1584 of SEQ ID NO:45,

nucleotides 1 to 1302 of SEQ ID NO:45,

nucleotides 1 to 1368 of SEQ ID NO:47,

nucleotides 1 to 1302 of SEQ ID NO:47,

nucleotides 1 to 1395 of SEQ ID NO:49,

nucleotides 1 to 1302 of SEQ ID NO:49,

nucleotides 1 to 1383 of SEQ ID NO:51,

nucleotides 1 to 1302 of SEQ ID NO:51,

nucleotides 1 to 1353 of SEQ ID NO:53,

nucleotides 1 to 1302 of SEQ ID NO:53,

nucleotides 1 to 1599 of SEQ ID NO:55,

nucleotides 1 to 1302 of SEQ ID NO:55,

nucleotides 1 to 1383 of SEQ ID NO:57,

nucleotides 1 to 1302 of SEQ ID NO:57,

nucleotides 1 to 1578 of SEQ ID NO:59,

nucleotides 1 to 1302 of SEQ ID NO:59,

nucleotides 1 to 1371 of SEQ ID NO:65, and

nucleotides 1 to 1302 of SEQ ID NO:65;

(ii) the complementary strand of the nucleotides selected from the groupconsisting of:

nucleotides 1 to 500 of SEQ ID NO:1,

nucleotides 1 to 500 of SEQ ID NO:3,

nucleotides 1 to 500 of SEQ ID NO:5,

nucleotides 1 to 500 of SEQ ID NO:7,

nucleotides 1 to 500 of SEQ ID NO:9,

nucleotides 1 to 500 of SEQ ID NO:11,

nucleotides 1 to 500 of SEQ ID NO:13,

nucleotides 1 to 500 of SEQ ID NO:15,

nucleotides 1 to 500 of SEQ ID NO:37,

nucleotides 1 to 500 of SEQ ID NO:39,

nucleotides 1 to 500 of SEQ ID NO:41,

nucleotides 1 to 500 of SEQ ID NO:43,

nucleotides 1 to 500 of SEQ ID NO:45,

nucleotides 1 to 500 of SEQ ID NO:47,

nucleotides 1 to 500 of SEQ ID NO:49,

nucleotides 1 to 500 of SEQ ID NO:51,

nucleotides 1 to 500 of SEQ ID NO:53,

nucleotides 1 to 500 of SEQ ID NO:55,

nucleotides 1 to 500 of SEQ ID NO:57,

nucleotides 1 to 500 of SEQ ID NO:59,

nucleotides 1 to 500 of SEQ ID NO:65,

nucleotides 1 to 221 of SEQ ID NO:17,

nucleotides 1 to 239 of SEQ ID NO:18,

nucleotides 1 to 199 of SEQ ID NO:19,

nucleotides 1 to 191 of SEQ ID NO:20,

nucleotides 1 to 232 of SEQ ID NO:21,

nucleotides 1 to 467 of SEQ ID NO:22,

nucleotides 1 to 534 of SEQ ID NO:23,

nucleotides 1 to 563 of SEQ ID NO:24,

nucleotides 1 to 218 of SEQ ID NO:25,

nucleotides 1 to 492 of SEQ ID NO:26,

nucleotides 1 to 481 of SEQ ID NO:27,

nucleotides 1 to 463 of SEQ ID NO:28,

nucleotides 1 to 513 of SEQ ID NO:29,

nucleotides 1 to 579 of SEQ ID NO:30,

nucleotides 1 to 514 of SEQ ID NO:31,

nucleotides 1 to 477 of SEQ ID NO:32,

nucleotides 1 to 500 of SEQ ID NO:33,

nucleotides 1 to 470 of SEQ ID NO:34,

nucleotides 1 to 491 of SEQ ID NO:35,

nucleotides 1 to 221 of SEQ ID NO:36,

nucleotides 1 to 519 of SEQ ID NO:61,

nucleotides 1 to 497 of SEQ ID NO:62,

nucleotides 1 to 498 of SEQ ID NO:63,

nucleotides 1 to 525 of SEQ ID NO:64, and

nucleotides 1 to 951 of SEQ ID NO:67; and

(iii) the complementary strand of the nucleotides selected from thegroup consisting of:

nucleotides 1 to 200 of SEQ ID NO:1,

nucleotides 1 to 200 of SEQ ID NO:3,

nucleotides 1 to 200 of SEQ ID NO:5,

nucleotides 1 to 200 of SEQ ID NO:7,

nucleotides 1 to 200 of SEQ ID NO:9,

nucleotides 1 to 200 of SEQ ID NO:11,

nucleotides 1 to 200 of SEQ ID NO:13,

nucleotides 1 to 200 of SEQ ID NO:15,

nucleotides 1 to 200 of SEQ ID NO:37,

nucleotides 1 to 200 of SEQ ID NO:39,

nucleotides 1 to 200 of SEQ ID NO:41,

nucleotides 1 to 200 of SEQ ID NO:43,

nucleotides 1 to 200 of SEQ ID NO:45,

nucleotides 1 to 200 of SEQ ID NO:47,

nucleotides 1 to 200 of SEQ ID NO:49,

nucleotides 1 to 200 of SEQ ID NO:51,

nucleotides 1 to 200 of SEQ ID NO:53,

nucleotides 1 to 200 of SEQ ID NO:55,

nucleotides 1 to 200 of SEQ ID NO:57,

nucleotides 1 to 200 of SEQ ID NO:59,

nucleotides 1 to 200 of SEQ ID NO:65,

nucleotides 1 to 200 of SEQ ID NO:22,

nucleotides 1 to 200 of SEQ ID NO:23,

nucleotides 1 to 200 of SEQ ID NO:24,

nucleotides 1 to 200 of SEQ ID NO:26,

nucleotides 1 to 200 of SEQ ID NO:27,

nucleotides 1 to 200 of SEQ ID NO:28,

nucleotides 1 to 200 of SEQ ID NO:29,

nucleotides 1 to 200 of SEQ ID NO:30,

nucleotides 1 to 200 of SEQ ID NO:31,

nucleotides 1 to 200 of SEQ ID NO:32,

nucleotides 1 to 200 of SEQ ID NO:33,

nucleotides 1 to 200 of SEQ ID NO:34,

nucleotides 1 to 200 of SEQ ID NO:35,

nucleotides 1 to 200 of SEQ ID NO:61,

nucleotides 1 to 200 of SEQ ID NO:62,

nucleotides 1 to 200 of SEQ ID NO:63,

nucleotides 1 to 200 of SEQ ID NO:64, and

nucleotides 1 to 200 of SEQ ID NO:67.

As will be understood, details and particulars concerning hybridizationof the nucleotide sequences will be the same or analogous to thehybridization aspects discussed in the section entitled “PolypeptidesHaving Cellobiohydrolase I Activity” herein.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleotide sequence of the present invention operably linked to one ormore control sequences that direct the expression of the coding sequencein a suitable host cell under conditions compatible with the controlsequences.

A nucleotide sequence encoding a polypeptide of the present inventionmay be manipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the nucleotide sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying nucleotide sequencesutilizing recombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofthe nucleotide sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of the polypeptide. Thepromoter may be any nucleotide sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising the nucleic acid construct of the invention. The variousnucleotide and control sequences described above may be joined togetherto produce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, the nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleotide sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.

The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof.

Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleotide sequence encoding the polypeptide or any other element of thevector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleotide sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleotide sequences enable the vector to be integrated intothe host cell genome at a precise location(s) in the chromosome(s). Toincrease the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleotides, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleotide sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a nucleotide sequence of the present invention maybe inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleotide sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleotide sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleotide sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant a host cell comprisingthe nucleic acid construct of the invention, which are advantageouslyused in the recombinant production of the polypeptides. A vectorcomprising a nucleotide sequence of the present invention is introducedinto a host cell so that the vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector as describedearlier.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In apreferred embodiment, the bacterial host cell is a Bacillus lentus,Bacillus licheniformis, Bacillus stearothermophilus, or Bacillussubtilis cell. In another preferred embodiment, the Bacillus cell is analkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823 -829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may be a eukaryote, such as a mammalian, insect, plant, orfungal cell.

In a preferred embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a more preferred embodiment, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred embodiment, the yeast host cell is a Candida,Aschbyii, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia cell.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred embodiment, the filamentous fungal host cellis a cell of a species of, but not limited to, Acremonium, Aspergillus,Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium,Thielavia, Tolypocladium, or Trichoderma.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal parent cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, 194: 182-187,Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide; and (b) recovering the polypeptide. Preferably, the strainis selected from the group consisting of Acremonium, Scytalidium,Thermoascus, Thielavia, Verticillium, Neotermes, Melanocarpus,Poitrasia, Coprinus, Trichothecium, Humicola, Cladorrhinum, Diplodia,Myceliophthora, Rhizomucor, Meripilus, Exidia, Xylaria, Trichophaea,Chaetomium, Chaetomidium, Sporotrichum, Thielavia, Aspergillus,Scopulariopsis, Fusarium, Pseudoplectania, and Phytophthora; morepreferably the strain is selected from the group consisting ofAcremonium thermophilum, Chaetomium thermophilum, Scytalidiumthermophilum, Thermoascus aurantiacus, Thielavia australiensis,Verticillium tenerum, Neotermes castaneus, Melanocarpus albomyces,Poitrasia circinans, Coprinus cinereus, Trichothecium roseum, Humicolanigrescens, Cladorrhinum foecundissimum, Diplodia gossypina,Myceliophthora thermophila, Rhizomucor pusillus, Meripilus giganteus,Exidia glandulosa, Xylaria hypoxylon, Trichophaea saccata, Chaetomidiumpingtungium, Myceliophthora thermophila, Myceliophthora hinnulea,Sporotrichum pruinosum, Thielavia cf. microspora, Pseudoplectanianigrella, and Phytophthora infestans.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for in-situ production ofa polypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) contacting the polypeptide with a desired substrate, such as acellulosic substrate, without prior recovery of the polypeptide. Theterm “in-situ production” is intended to mean that the polypeptide isproduced directly in the locus in which it is intended to be used, suchas in a fermentation process for production of ethanol.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having cellobiohydrolase I activity of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing therecombinant polypeptide may be used as such for improving the quality ofa food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, millets, and maize (corn).

Examples of dicot plants are tobacco, lupins, potato, sugar beet,legumes, such as pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape, canola, and the closelyrelated model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers. Also specific plant tissues, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part.

Also included within the scope of the present invention are the progeny(clonal or seed) of such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleotide sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV promoter may be used (Francket al., 1980, Cell 21: 285-294). Organ-specific promoters may be, forexample, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588).

A promoter enhancer element may also be used to achieve higherexpression of the enzyme in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleotide sequenceencoding a polypeptide having cellobiohydrolase I activity of thepresent invention under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods for in-situ production ofa polypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleotide sequenceencoding a polypeptide having cellobiohydrolase I activity of thepresent invention under conditions conducive for production of thepolypeptide; and (b) contacting the polypeptide with a desiredsubstrate, such as a cellulosic substrate, without prior recovery of thepolypeptide.

Compositions

In a still further aspect, the present invention relates to compositionscomprising a polypeptide of the present invention.

The composition may comprise a polypeptide of the invention as the majorenzymatic component, e.g., a mono-component composition. Alternatively,the composition may comprise multiple enzymatic activities, such as anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase,invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase, or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Forinstance, the polypeptide composition may be in the form of a granulateor a microgranulate. The polypeptide to be included in the compositionmay be stabilized in accordance with methods known in the art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Detergent Compositions

The polypeptide of the invention may be added to and thus become acomponent of a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the polypeptide of the invention. The detergent additive aswell as the detergent composition may comprise one or more other enzymessuch as a protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta,1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of B. licheniformis,described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated e.g., as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238 216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g., WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the polypeptide of the invention, may be added inan amount corresponding to 0.01-100 mg of enzyme protein per liter ofwash liquor, preferably 0.05-5 mg of enzyme protein per liter of washliquor, in particular 0.1-1 mg of enzyme protein per liter of washliquor.

The polypeptide of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202 which is herebyincorporated as reference.

DNA Recombination (Shuffling)

The nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,

SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62,SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67 may be used in aDNA recombination (or shuffling) process. The new polynucleotidesequences obtained in such a process may encode new polypeptides havingcellobiase activity with improved properties, such as improved stability(storage stability, thermostability), improved specific activity,improved pH-optimum, and/or improved tolerance towards specificcompounds.

Shuffling between two or more homologous input polynucleotides(starting-point polynucleotides) involves fragmenting thepolynucleotides and recombining the fragments, to obtain outputpolynucleotides (i.e., polynucleotides that have been subjected to ashuffling cycle) wherein a number of nucleotide fragments are exchangedin comparison to the input polynucleotides.

DNA recombination or shuffling may be a (partially) random process inwhich a library of chimeric genes is generated from two or more startinggenes. A number of known formats can be used to carry out this shufflingor recombination process.

The process may involve random fragmentation of parental DNA followed byreassembly by PCR to new full-length genes, e.g., as presented in U.S.Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 6,117,679. In-vitrorecombination of genes may be carried out, e.g., as described in U.S.Pat. Nos. 6,159,687, 6,159,688, 5,965,408, 6,153,510, and WO 98/41623.The recombination process may take place in vivo in a living cell, e.g.,as described in WO 97/07205 and WO 98/28416.

The parental DNA may be fragmented by DNA'se I treatment or byrestriction endonuclease digests as described by Kikuchi et al (2000a,Gene 236:159-167). Shuffling of two parents may be done by shufflingsingle stranded parental DNA of the two parents as described in Kikuchiet al (2000b, Gene 243:133-137).

A particular method of shuffling is to follow the methods described inCrameri et al, 1998, Nature 391: 288-291 and Ness et al., NatureBiotechnology 17: 893-896. Another format would be the methods describedin U.S. Pat. No. 6,159,687: Examples 1 and 2.

Production of Ethanol from Biomass

The present invention also relates to methods for producing ethanol frombiomass, such as cellulosic materials, comprising contacting the biomasswith the polypeptides of the invention. Ethanol may subsequently berecovered. The polypeptides of the invention may be produced “in-situ”,i.e., as part of, or directly in an ethanol production process, bycultivating a host cell or a strain, which in its wild-type form iscapable of producing the polypeptides, under conditions conducive forproduction of the polypeptides.

Ethanol can be produced by enzymatic degradation of biomass andconversion of the released polysaccharides to ethanol. This kind ofethanol is often referred to as bioethanol or biofuel. It can be used asa fuel additive or extender in blends of from less than 1% and up to100% (a fuel substitute). In some countries, such as Brazil, ethanol issubstituting gasoline to a very large extent.

The predominant polysaccharide in the primary cell wall of biomass iscellulose, the second most abundant is hemi-cellulose, and the third ispectin. The secondary cell wall, produced after the cell has stoppedgrowing, also contains polysaccharides and is strengthened throughpolymeric lignin covalently cross-linked to hemicellulose. Cellulose isa homopolymer of anhydrocellobiose and thus a linearbeta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which helps stabilize the cell wall matrix.

Three major classes of cellulase enzymes are used to breakdown biomass:

The “endo-1,4-beta-glucanases” or 1,4-beta-D-glucan-4-glucanohydrolases(EC 3.2.1.4), which act randomly on soluble and insoluble1,4-beta-glucan substrates.

The “exo-1,4-beta-D-glucanases” including both the 1,4-beta-D-glucanglucohydrolases (EC 3.2.1.74), which liberate D-glucose from1,4-beta-D-glucans and hydrolyze D-cellobiose slowly, and1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91), also referred to ascellobiohydrolase I, which liberates D-cellobiose from 1,4-beta-glucans.

The “beta-D-glucosidases” or beta-D-glucoside glucohydrolases (EC3.2.1.21), which act to release D-glucose units from cellobiose andsoluble cellodextrins, as well as an array of glycosides.

These three classes of enzymes work together synergistically in acomplex interplay that results in efficient decrystallization andhydrolysis of native cellulose from biomass to yield the reducing sugarswhich are converted to ethanol by fermentation.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Example 1 Cloning of a Partial and a Full-Length Cellobiohydrolase I(CBH1) DNA Sequence

A cDNA library of Diplodia gossypina was PCR screened for presence ofthe CBH1 gene. For this purpose sets of primers were constructed, basedon sequence alignment and identification of conserved regions among CBH1proteins. The PCR band from a gel electrophoresis was used to obtain apartial sequence of the CBH1 gene from Diplodia gossypina. Homologysearch confirmed that the partial sequence was a partial sequence of theCBH1 gene (EC 3.2.1.91).

The full-length CBH1 gene of Diplodia gossypina is obtained by accessingthe patent deposit CBS 247.96, make a DNA or cDNA preparation, use thepartial sequence as basis for construction of specific primers, and usestandard PCR cloning techniques to step by step getting the entire gene.

Several other approaches can be taken:

PCR screening of the cDNA library or the cDNAs that were used for theconstruction of the library, could be performed. To do so, Gene SpecificPrimers (GSP) and vector/adaptor primers are constructed from thepartial cDNA sequence of the CBH1 gene and from vector/adaptor sequencerespectively; both sets of primers designed to go outward into themissing 5′ and 3′ regions of the CBH1 cDNA. The longest PCR productsobtained using combinations of GSP and vector/adaptor primer representthe full-length 5′ and 3′ end regions of the CBH1 cDNA from Diplodiagossypina. Homology search and comparison with the partial cDNA sequenceconfirm that the 5′ and 3′ PCR products belong to the same CBH1 cDNAfrom Diplodia gossypina. The full-length cDNA can then be obtained byPCR using a set of primers constructed from both the 5′ and 3′ ends.

Alternatively, the cDNA library could be screened for the full-lengthcDNA using standard hybridization techniques and the partial cDNAsequence as a probe. The clones giving a positive hybridization signalwith the probe are then purified and sequenced to determine the longestcDNA sequence. Homology search and comparison confirms that thefull-length cDNA correspond to the partial CBH1 cDNA sequence that wasoriginally used as a probe.

The two approaches described above rely on the presence of thefull-length CBH1 cDNA in the cDNA library or in the cDNAs used for itsconstruction. Alternatively, the 5′ and 3′ RACE (Rapid Amplification ofcDNA Ends) techniques or derived techniques could be used to identifythe missing 5′ and 3′ regions. For this purpose, preferably mRNAs fromDiplodia gossipina are isolated and utilized to synthesize first strandcDNAs using oligo(dT)-containing Adapter Primer or a 5′-Gene SpecificPrimer (GSP).

The full-length cDNA of the CBH1 gene from Diplodia gossypina can alsobe obtained by using genomic DNA from Diplodia gossypina. The CBH1 genecan be identified by PCR techniques such as the one describe above or bystandard genomic library screening using hybridization techniques andthe partial CBH1 cDNA as a probe. Homology search and comparison withthe partial CBH1 cDNA confirms that the genomic sequence correspond tothe CBH1 gene from Diplodia gossypina. Identification of consensussequences such as initiation site of transcription, start and stopcodons or polyA sites could be used to define the region comprising thefull-length cDNA. Primers constructed from both the 5′ and 3′ ends ofthis region could then be used to amplify the full-length cDNA from mRNAor cDNA library from Diplodia gossypina (see above).

By expression of the full-length gene in a suitable expression hostconstruct the CBH1 enzyme is harvested as an intra cellular or extracellular enzyme from the culture broth.

The methods described above apply to the cloning of cellobiohydrolase IDNA sequences from all organisms and not only Diplodia gossypina.

Example 2 Cellobiohydrolase I (CBH I) Activity

A cellobiohydrolase I is characterized by the ability to hydrolyzehighly crystalline cellulose very efficiently compared to othercellulases. Cellobiohydrolase I may have a higher catalytic activityusing PASO (phosphoric acid swollen cellulose) as substrate than usingCMC as substrate. For the purposes of the present invention, any of thefollowing assays can be used to identify a cellobiohydrolase I:

Activity on Azo-Avicel

Azo-Avicel (Megazyme, Bray Business Park, Bray, Wicklow, Ireland) wasused according to the manufacturer's instructions.

Activity on PNP-Beta-Cellobiose

-   Substrate solution: 5 mM PNP beta-D-Cellobiose (p-Nitrophenyl    β-d-Cellobioside Sigma N-5759) in 0.1 M Na-acetate buffer, pH 5.0;-   Stop reagent: 0.1 M Na-carbonate, pH 11.5.

50 microliters CBH I solution was mixed with 1 mL substrate solution andincubated 20 minutes at 40° C. The reaction was stopped by addition of 5mL stop reagent. Absorbance was measured at 404 nm.

Activity on PASO and CMC

The substrate is degraded with cellobiohydrolase I (CBH I) to formreducing sugars. A Microdochium nivale carbohydrate oxidase (rMnO) oranother equivalent oxidase acts on the reducing sugars to form H₂O₂ inthe presence of O₂. The formed H₂O₂ activates in the presence of excessperoxidase the oxidative condensation of 4-aminoantipyrine (AA) andN-ethyl-N-sulfopropyl-m-toluidine (TOPS) to form a purple product whichcan be quantified by its absorbance at 550 nm.

When all components except CBH I are in surplus, the rate of increase inabsorbance is proportional to the CBH I activity. The reaction is aone-kinetic-step reaction and may be carried out automatically in aCobas Fara centrifugal analyzer (Hoffmann La Roche) or anotherequivalent spectrophotometer which can measure steady state kinetics.

-   Buffer: 50 mM Na-acetate buffer (pH 5.0);-   Reagents: rMnO oxidase, purified Microdochium nivale carbohydrate    oxidase, 2 mg/L (final concentration);    -   Peroxidase, SIGMA P-8125 (96 U/mg), 25 mg/L (final        concentration);    -   4-aminoantipyrine, SIGMA A-4382, 200 mg/L (final concentration);    -   TOPS, SIGMA E-8506, 600 mg/L (final concentration);    -   PASO or CMC (see below), 5 g/L (final concentration).

All reagents were added to the buffer in the concentrations indicatedabove and this reagent solution was mixed thoroughly.

50 microliters cellobiohydrolase I sample (in a suitable dilution) wasmixed with 300 μL reagent solution and incubated 20 minutes at 40° C.Purple color formation was detected and measured as absorbance at 550nm.

The AA/TOPS-condensate absorption coefficient is 0.01935 A₅₅₀/(microMcm). The rate is calculated as micromoles reducing sugar produced perminute from OD₅₅₀/minute and the absorption coefficient.

PASC:

-   Materials: 5 g Avicel® (Art. 2331 Merck);    -   150 mL 85% Ortho-phosphoric-acid (Art. 573 Merck);    -   800 mL Acetone (Art. 14 Merck);    -   Approx. 2 liter deionized water (Milli-Q);    -   1 L glass beaker;    -   1 L glass filter funnel;    -   2 L suction flask;    -   Ultra Turrax Homogenizer.

Acetone and ortho-phosphoric-acid is cooled on ice. Avicel® is moistedwith water, and then the 150 mL icecold 85% Ortho-phosphoric-acid isadded. The mixture is placed on an icebath with weak stirring for onehour.

Add 500 mL ice-cold acetone with stirring, and transfer the mixture to aglass filter funnel and wash with 3×100 mL ice-cold acetone, suck as dryas possible in each wash. Wash with 2×500 mL water (or until there is noodor of acetone), suck as dry as possible in each wash.

Re-suspend the solids in water to a total volume of 500 mL, and blend tohomogeneity using an Ultra Turrax Homogenizer. Store wet in refrigeratorand equilibrate with buffer by centrifugation and re-suspension beforeuse.

CMC:

Bacterial cellulose microfibrils in an impure form were obtained fromthe Japanese foodstuff “nata de coco” (Fujico Company, Japan). Thecellulose in 350 g of this product was purified by suspension of theproduct in about 4 L of tap water. This water was replaced by freshwater twice a day for 4 days.

Then 1% (w/v) NaOH was used instead of water and the product wasre-suspended in the alkali solution twice a day for 4 days.Neutralisation was done by rinsing the purified cellulose with distilledwater until the pH at the surface of the product was neutral (pH 7).

The cellulose was microfibrillated and a suspension of individualbacterial cellulose microfibrils was obtained by homogenisation of thepurified cellulose microfibrils in a Waring blender for 30 min. Thecellulose microfibrils were further purified by dialysing thissuspension through a pore membrane against distilled water and theisolated and purified cellulose microfibrils were stored in a watersuspension at 4° C.

Deposit of Biological Material China General Microbiological CultureCollection Center (CGMCC)

The following biological material has been deposited under the terms ofthe Budapest Treaty with the China General Microbiological CultureCollection Center (CGMCC), Institute of Microbiology, Chinese Academy ofSciences, Haidian, Beijing 100080, China:

-   Accession Number: CGMCC No. 0584-   Applicants reference: ND000575-   Date of Deposit: 2001-05-29-   Description: Acremonium thermophilum CBH I gene on plasmid-   Classification: Ascomycota; Sordariomycetes; Hypocrales;    Hypocreaceae-   Origin: China, 1999-   Related sequence(s): SEQ ID NO:1 and SEQ ID NO:2 (DNA sequence    encoding a cellobiohydrolase I from Acremonium thermophilum and the    corresponding protein sequence)-   Accession Number: CGMCC No. 0581-   Applicants reference: ND000548-   Date of Deposit: 2001-05-29-   Description: Chaetomium thermophilum CBH I gene on plasmid-   Classification: Ascomycota; Sordariomycetes; Sordariales;    Chaetomiaceae-   Origin: China, 1999-   Related sequence(s): SEQ ID NO:3 and SEQ ID NO:4 (DNA sequence    encoding a cellobiohydrolase I from Chaetomium thermophilum and the    corresponding protein sequence)-   Accession Number: CGMCC No. 0585-   Applicants reference: ND001223-   Date of Deposit: 2001-05-29-   Description: Scytalidium sp. CBH I gene on plasmid-   Classification: Ascomycota; Mitosporic-   Origin: China, 1999-   Related sequence(s): SEQ ID NO:5 and SEQ ID NO:6 (DNA sequence    encoding a cellobiohydrolase I from Scytalidium sp. and the    corresponding protein sequence)-   Accession Number: CGMCC No. 0582-   Applicants reference: ND000549-   Date of Deposit: 2001-05-29-   Description: Thermoascus aurantiacus CBH I gene on plasmid-   Classification: Eurotiomycetes; Eurotiales; Trichocomaceae-   Origin: China-   Related sequence(s): SEQ ID NO:7 and SEQ ID NO:8 (DNA sequence    encoding a cellobiohydrolase I from Thermoascus aurantiacus and the    corresponding protein sequence)-   Accession Number: CGMCC No. 0583-   Applicants reference: ND001182-   Date of Deposit: 2001-05-29-   Description: Thielavia australiensis CBH I gene on plasmid-   Classification: Ascomycota; Sordariomycetes; Sordariales;    Chaetomiaceae-   Origin: China, 1998-   Related sequence(s): SEQ ID NO:9 and SEQ ID NO:10 (DNA sequence    encoding a cellobiohydrolase I from Thielavia australiensis and the    corresponding protein sequence)-   Accession Number: CGMCC No. 0580-   Applicants reference: ND000562-   Date of Deposit: 2001-05-29-   Description: Melanocarpus albomyces CBH I gene on plasmid-   Classification: Ascomycota; Sordariomycetes; Sordariales-   Origin: China, 1999-   Related sequence(s): SEQ ID NO:15 and SEQ ID NO:16 (DNA sequence    encoding a cellobiohydrolase I from Melanocarpus albomyces and the    corresponding protein sequence)-   Accession Number: CGMCC No. 0748-   Applicants reference: ND001181-   Date of Deposit: 2002-06-07-   Description: Acremonium sp. CBH I gene on plasmid-   Classification: mitosporic Ascomycetes-   Origin: China, 2000-   Related sequence(s): SEQ ID NO:53 and SEQ ID NO:54-   Accession Number: CGMCC No. 0749-   Applicants reference: ND000577-   Date of Deposit: 2002-06-07-   Description: Aspergillus fumigatus CBH I gene on plasmid-   Classification: Trichocomaceae, Eurotiales, Ascomycota (Teleomorph:    Neosartorya fumigata)-   Origin: China, 2000-   Related sequence(s): SEQ ID NO:55 and SEQ ID NO:56-   Accession Number: CGMCC No. 0747-   Applicants reference: ND001175-   Date of Deposit: 2002-06-07-   Description: Sporotrichum pruinosum CBH I gene on plasmid-   Classification: Meruliaceae, Stereales, Basidiomycota-   Origin: China, 2000-   Related sequence(s): SEQ ID NO:57 and SEQ ID NO:58-   Accession Number: CGMCC No. 0750-   Applicants reference: ND000571-   Date of Deposit: 2002-06-07-   Description: Scytalidium thermophilum CBH I gene on plasmid-   Classification: Ascomycota; Mitosporic-   Origin: China, 2000-   Related sequence(s): SEQ ID NO:59 and SEQ ID NO:60

Centraalbureau Voor Schimmelcultures (CBS)

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Centraalbureau Voor Schimmelcultures (CBS),Uppsalalaan 8, 3584 CT Utrecht, The Netherlands (alternatively P.O. Box85167, 3508 AD Utrecht, The Netherlands):

-   Accession Number: CBS109513-   Applicants reference: ND000538-   Date of Deposit: 2001-06-01-   Description: Verticillium tenerum-   Classification: Ascomycota, Hypocreales, Pyrenomycetes (mitosporic)-   Origin: —-   Related sequence(s): SEQ ID NO:11 and SEQ ID NO:12 (DNA sequence    encoding a cellobiohydrolase I from Verticillium tenerum and the    corresponding protein sequence)-   Accession Number: CBS 819.73-   Applicants reference: ND000533-   Date of Deposit: Publicly available (not deposited by applicant)-   Description: Humicola nigrescens-   Classification: Sordariaceae, Sordariales, Sordariomycetes;    Ascomycota-   Origin: —-   Related sequence(s): SEQ ID NO:18 (partial DNA sequence encoding a    cellobiohydrolase I from Humicola nigrescens)-   Accession Number: CBS 427.97-   Applicants reference: ND000530-   Date of Deposit: 1997-01-23-   Description: Cladorrhinum foecundissimum-   Classification: Sordariaceae, Sordariales, Sordariomycetes;    Ascomycota-   Origin: Jamaica-   Related sequence(s): SEQ ID NO:19 (partial DNA sequence encoding a    cellobiohydrolase I from Cladorrhinum foecundissimum)-   Accession Number: CBS 247.96-   Applicants reference: ND000534 and ND001231-   Date of Deposit: 1996-03-12-   Description: Diplodia gossypina-   Classification: Dothideaceae, Dothideales, Dothidemycetes;    Ascomycota-   Origin: Indonesia, 1992-   Related sequence(s): SEQ ID NO:20 (partial DNA sequence encoding a    cellobiohydrolase I from Diplodia gossypina), SEQ ID NO:37 (full DNA    sequence encoding a cellobiohydrolase I from Diplodia gossypina) and    SEQ ID NO:38 (full cellobiohydrolase I protein sequence from    Diplodia gossypina)-   Accession Number: CBS117.65-   Applicants reference: ND000536-   Date of Deposit: Publicly available-   Description: Myceliophthora thermophila-   Classification: Sordariaceae, Sordariales, Sordariomycetes;    Ascomycota-   Origin: —-   Related sequence(s): SEQ ID NO:21 (partial DNA sequence encoding a    cellobiohydrolase I from Myceliophthora thermophila)-   Accession Number: CBS109471-   Applicants reference: ND000537-   Date of Deposit: 2001-05-29-   Description: Rhizomucor pusillus-   Classification: Mucoraceae, Mucorales, Zygomycota-   Origin: Denmark-   Related sequence(s): SEQ ID NO:22 (partial DNA sequence encoding a    cellobiohydrolase I from Rhizomucor pusillus)-   Accession Number: CBS 521.95-   Applicants reference: ND000542-   Date of Deposit: 1995-07-04-   Description: Meripilus giganteus-   Classification: Rigidiporaceae, Hymenomycetales, Basidiomycota-   Origin: Denmark, 1993-   Related sequence(s): SEQ ID NO:23 (partial DNA sequence encoding a    cellobiohydrolase I from Meripilus giganteus)-   Accession Number: CBS 277.96-   Applicants reference: ND000543, ND001346 and ND001243-   Date of Deposit: 1996-03-12-   Description: Exidia glandulosa-   Classification: Exidiaceae, Auriculariales, Hymenomycetes,    Basidiomycota-   Origin: Denmark, 1993-   Related sequence(s): SEQ ID NO:24 (partial DNA sequence encoding a    cellobiohydrolase I from Exidia glandulosa), SEQ ID NO:45 (full DNA    sequence encoding a cellobiohydrolase I with CBD from Exidia    glandulosa), SEQ ID NO:46 (full cellobiohydrolase I protein sequence    with CBD from Exidia glandulosa), SEQ ID NO:47 (full DNA sequence    encoding a cellobiohydrolase I from Exidia glandulosa) and SEQ ID    NO:48 (full cellobiohydrolase I protein sequence from Exidia    glandulosa)-   Accession Number: CBS 284.96-   Applicants reference: ND000544 and ND001235-   Date of Deposit: 1996-03-12-   Description: Xylaria hypoxylon-   Classification: Sordariaceae, Sordariales, Sordariomycetes,    Ascomycota-   Origin: Denmark, 1993-   Related sequence(s): SEQ ID NO:25 (partial DNA sequence encoding a    cellobiohydrolase I from Xylaria hypoxylon), SEQ ID NO:43 (full DNA    sequence encoding a cellobiohydrolase I from Xylaria hypoxylon) and    SEQ ID NO:44 (full cellobiohydrolase I protein sequence from Xylaria    hypoxylon)-   Accession Number: CBS 804.70-   Applicants reference: ND001227-   Date of Deposit: Publicly available-   Description: Trichophaea saccata-   Classification: Ascomycota; Pezizomycetes; Pezizales; Pyronemataceae-   Related sequence(s): SEQ ID NO:36 (partial DNA sequence encoding a    cellobiohydrolase I from Trichophaea saccata)

Deutsche Sammlunq von Mikroorganismen und Zellkulturen GmbH (DSMZ)

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Mascheroder Weg 1b, 38124 Braunschweig,Germany:

-   Accession Number: DSM 14348-   Applicants reference: ND000551-   Date of Deposit: 2001-06-13-   Description: Neotermes castaneus, termite CBH I gene on plasmid-   Classification: —-   Origin: Cultures of termite larvae bought from BAM, Germany, 1999-   Related sequence(s): SEQ ID NO:13 and SEQ ID NO:14 (DNA sequence    encoding a cellobiohydrolase I from gut cells or microbes from the    gut of Neotermes castaneus and the corresponding protein sequence)-   Accession Number: DSM 15066-   Applicants reference: ND001349-   Date of Deposit: 2002-06-21-   Description: Poitrasia circinans CBH I gene on plasmid-   Classification: Choanephoraceae, Zygomycota, Mucorales-   Origin: —-   Related sequence(s): SEQ ID NO:49 (DNA sequence encoding a    cellobiohydrolase I from Poitrasia circinans) and SEQ ID NO:50    (cellobiohydrolase I protein sequence from Poitrasia circinans)-   Accession Number: DSM 15065-   Applicants reference: ND001339-   Date of Deposit: 2002-06-21-   Description: Coprinus cinereus CBH I gene on plasmid-   Classification: Basidiomycota, Hymenomycetes; Agaricales,    Agaricaceae-   Origin: Denmark-   Related sequence(s): SEQ ID NO:51 (DNA sequence encoding a    cellobiohydrolase I from Coprinus cinereus) and SEQ ID NO:52    (cellobiohydrolase I protein sequence from Coprinus cinereus)-   Accession Number: DSM 15064-   Applicants reference: ND001264-   Date of Deposit: 2002-06-21-   Description: Trichophaea saccata CBH I gene on plasmid-   Classification: Ascomycota; Pezizomycetes; Pezizales; Pyronemataceae-   Origin: —-   Related sequence(s): SEQ ID NO:39 (DNA sequence encoding a    cellobiohydrolase I from Trichophaea saccata) and SEQ ID NO:40    (cellobiohydrolase I protein sequence from Trichophaea saccata)-   Accession Number: DSM 15067-   Applicants reference: ND001232-   Date of Deposit: 2002-06-21-   Description: Myceliophthora thermophila CBH I gene on plasmid-   Classification: Sordariaceae, Sordariales, Sordariomycetes;    Ascomycota-   Origin: —-   Related sequence(s): SEQ ID NO:41 (DNA sequence encoding a    cellobiohydrolase I from Myceliophthora thermophila) and SEQ ID    NO:42 (cellobiohydrolase I protein sequence from Myceliophthora    thermophila)

Institute for Fermentation, Osaka (IFO)

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Institute for Fermentation, Osaka (IFO),17-85, Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan:

-   Accession Number: IFO 5372-   Applicants reference: ND000531-   Date of Deposit: Publicly available (not deposited by applicant)-   Description: Trichothecium roseum-   Classification: mitosporic Ascomycetes-   Origin: —-   Related sequence(s): SEQ ID NO:17 (partial DNA sequence encoding a    cellobiohydrolase I from Trichothecium roseum)

The deposit of CBS 427.97, CBS 247.96, CBS 521.95, CBS 284.96, CBS274.96 were made by Novo Nordisk A/S and were later assigned toNovozymes A/S.

1-29. (canceled)
 30. An isolated polypeptide having cellobiohydrolase Iactivity, which has at least 80% identity with the sequence of aminoacids 1 to 529 of SEQ ID NO:4.
 31. The polypeptide of claim 30, whichhas at least 85% identity with amino acids 1 to 529 of SEQ ID NO:4. 32.The polypeptide of claim 30, which has at least 90% identity with aminoacids 1 to 529 of SEQ ID NO:4.
 33. The polypeptide of claim 30, whichhas at least 95% identity with amino acids 1 to 529 of SEQ ID NO:4. 34.The polypeptide of claim 30, which has at least 97% identity with aminoacids 1 to 529 of SEQ ID NO:4.
 35. The polypeptide of claim 30, whichhas at least 98% identity with amino acids 1 to 529 of SEQ ID NO:4. 36.The polypeptide of claim 30, which has at least 99% identity with aminoacids 1 to 529 of SEQ ID NO:4.
 37. The polypeptide of claim 30,comprising the sequence of amino acids 1 to 529 of SEQ ID NO:4.
 38. Thepolypeptide of claim 30, which is a fragment of the sequence of aminoacids 1 to 529 of SEQ ID NO:4.
 39. A detergent composition comprising asurfactant and the polypeptide of claim
 30. 40. A method for producingethanol from biomass, comprising (a) contacting the biomass with thepolypeptide of claim 30, an endo-1,4-beta-glucanase, and abeta-D-glucosidase to produce sugar; and (b) fermenting the sugar toproduce ethanol.